Methods for the production of apolipoproteins in transgenic plants

ABSTRACT

Methods for the production of an apolipoprotein in plants are described. In one embodiment, the present invention provides a method for the expression of apolipoprotein in plants comprising:
         (a) providing a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of transcription as operably linked components:
           (i) a nucleic acid sequence capable of controlling expression in plant cells; and   (ii) a nucleic acid sequence encoding an apolipoprotein polypeptide;   
           (b) introducing the chimeric nucleic acid construct into a plant cell; and
 
growing the plant cell into a mature plant capable of setting seed wherein the seed expresses apolipoprotein.

FIELD OF THE INVENTION

The present invention relates to plant genetic engineering methods andto the production of apolipoproteins. More specifically, the presentinvention relates to methods for the production of recombinantapolipoproteins in transgenic plants.

BACKGROUND OF THE INVENTION

In a healthy human body, there is a balance between the delivery andremoval of cholesterol. When people have a high level of low-densitylipoprotein (LDL) and low level of high-density lipoprotein (HDL), theimbalance results in more cholesterol being deposited in the arteriesthan that being removed (van Dam, M. J. et al. 2002, Lancet 359:37-42)). Atherosclerosis, the narrowing or blocking of arteries, is aconsequence of the repeated deposit of cholesterol, termed plaque(Major, A. S. et al. 2001, Arterioscler. Thromb. Vasc. Biol. 21:1790-1795)).

Lipoproteins can be separated into atherogenetic and vasoprotectivelipoproteins. Atherogenetic lipoproteins are generally allapolipoprotein (Apo) B-containing lipoprotein such as very-low-densitylipoprotein (VLDL), intermediate (IDL), low (LDL) or lipoprotein(Lp(a)), whereas vasoprotective lipoproteins are Apo AI containing, suchas HDL.

Apo AI, the major protein constituent of HDL, plays a critical role incholesterol homeostasis. Clinical and population-based studies havedemonstrated a remarkable inverse correlation between cardiovasculardisease and plasma HDL levels, suggesting Apo AI and HDL help to serve aprotective role against atherogenesis (Rubins, H. B. et al. 1993, Am. J.Cartiol. 71: 45-52)). Studies of transgenic mice (Rubin, E. M. et al.1991, Nature 353: 265-267)) and rabbits (Duverger, N. et al. 1996,Circulation 94: 713-717)) susceptible to atherosclerosis have shown thatexpression of human Apo AI inhibits the development of atherosclerosis.This effect may be related to its efficient promotion of cholesterolefflux from cells (Castro, G. et al. 1997, Biochemistry 36: 2243-2249)),the first step in the process of ‘reverse cholesterol transport’ (RCT)(Glomset, J. A. 1968, J Lipid Res. 9: 155-167)). Apo AI modulates thisprocess by being a preferential acceptor of cellular cholesterol(Rothblat, G. H. et al. 1999, J Lipid Res. 40: 781-796)), increasing theactivity of lecithin-cholesterol-acyl-transferase (LCAT) esterificationof HDL-associated cholesterol several-fold (Jonas, A. 1991, Biochim.Biophys. Acta 1084: 205-220; Mahley, R. W. et al. 1984, J Lipid Res. 25:1277-1294)), and transporting LCAT-derived cholesteryl esters to theliver (Morrison, J. R. et al. 1992, J Biol. Chem. 267: 13205-13209)).

Unlike synthetic antihyperlipidemics, such as LIPITOR® (atorvastatincalcium) that act to lower lipid levels in the body by inhibiting thesynthesis of cholesterol (Alaupovic, P. et al. 1997, Atherosclerosis133: 123-133)), an infusion of purified Apo AI stimulates cholesterolefflux from tissues into plasma (Navab, M. et al. 2002, Circulation 105:290-292)). This suggests that Apo AI could stimulate cholesterol effluxfrom foam cells in the arterial wall and induce regression ofatherosclerotic plaque, effectively ‘cleaning out’ the arteries.

In humans, Apo AI is synthesized in liver and intestinal cells as anon-glycosylated pre-pro-protein (Gordon, J. I. et al. 1983, J. Biol.Chem. 258: 4037-4044)). The 18 amino acid pre-segment is removed beforethe protein leaves the cell and the 6 amino acid pro-segment is cleavedpost secretion by an unknown protease in the plasma, leaving the mature243 amino acid protein (Saku, K. et al. 1999, Eur. J. Clin. Invest. 29:196-203)).

The Apolipoprotein A-I_(Milano) (Apo AI-M) is the first describedmolecular variant of human Apo AI (Franceschini, G. et al. 1980, J.Clin. Invest. 66: 892-900)). It is characterized by the substitution ofArg 173 with Cys (Weisgraber, K. H. et al. 1983, J. Biol. Chem. 258:2508-2513)). The mutant apolipoprotein is transmitted as an autosomaldominant trait and 8 generations of carriers have been identified(Gerli, G. C. et al. 1984, Hum. Hered. 34: 133-140)).

The status of the Apo AI-M carrier individual is characterized by aremarkable reduction in HDL-cholesterol level. In spite of this, theaffected subjects do not apparently show any increased risk of arterialdisease; indeed, by examination of the genealogic tree it appears thatthese subjects may be “protected” from atherosclerosis.

The mechanism of the possible protective effect of Apo AI-M in thecarriers seems to be linked to a modification in the structure of themutant apolipoprotein, with the loss of one alpha-helix and an increasedexposure of hydrophobic residues (Franceschini, G. et al. 1985, J. Biol.Chem. 260: 16321-16325). The loss of the tight structure of the multiplealpha-helices leads to an increased flexibility of the molecule, whichassociates more readily with lipids, compared to normal A-I. Moreover,apolipoprotein/lipid complexes are more susceptible to denaturation,thus suggesting that lipid delivery is also improved in the case of themutant.

The therapeutic use of Apo AI and the Apo AI-M mutant is presentlylimited by the lack of a method allowing the preparation of saidapolipoproteins in sufficient amount and in a suitable form. Inparticular, the recombinant production of Apo AI has been shown to bevery difficult due to its amphiphilic character, autoaggregation, anddegradation (Schmidt, H. H. et al. 1997, Protein Expr. Purif. 10:226-236). At the time of the present invention, recombinant human Apo AIhas been expressed in vitro in two eukaryotic systems: Baculovirustransfected Spodoptera frugiperda (Sf9) cells (Sorci-Thomas, M. G. etal. 1996, J. Lipid Res. 37: 673-683) and stably transfected Chinesehamster ovary (CHO) cells (Forte, T. M. et al. 1990, Biochim. Biophys.Acta 1047: 11-18; Mallory, J. B. et al. 1987, J. Biol. Chem. 262:4241-4247). In the baculovirus system, once the cells are successfullytransfected, there is an in-depth screening process before cells withthe correct construct can be used for expression. Similarly, CHO cellcolonies must undergo a screening process to find stably transfected,high expressing colonies. Additionally, both of these cell types requirea relatively long period of time before significant expression isachieved and a much higher level of maintenance than bacteria.

Recombinant expression of proteins in bacterial systems is generallyattractive because it can produce large amounts of pure protein quicklyand economically There are several reports of Apo AI expression intransformed Escherichia coli (E. coli); however, while certain recentimprovements in expression levels have been made (Ryan, R. O. et al.2003, Protein Expr. Purif. 27: 98-103) in general, these methods providerelatively low yields or the undesirable presence of extraneous affinitytags or secretion signals (Bergeron, J. et al. 1997, Biochim. Biophys.Acta 1344: 139-152; Li, H. H. et al. 2001, J. Lipid Res. 42: 2084-2091;McGuire, K. A. et al. 1996, J. Lipid Res. 37: 1519-1528). Moreover, E.coli endotoxins are known to form particularly strong complexes withapolipoproteins (Emancipator et al. (1992) Infect. Immun. 60: 596-601).Reduction or elimination of the toxicity associated with these E. coliendotoxins in pharmaceutical preparations of apoliproteins is highlydesirable. The removal of these endotoxins, while technically feasible,involves complex and expensive protein purification methodologies (U.S.Pat. No. 6,506,879) without fully eliminating the human health risk.

The use of plants as bioreactors for the large scale production ofrecombinant proteins is known to the art, and numerous proteins,including human therapeutic proteins, have been produced. For example,U.S. Pat. Nos. 4,956,282, 5,550,038 and 5,629,175 disclose theproduction of γ-interferon in plants; U.S. Pat. Nos. 5,650,307,5,716,802 and 5,763,748 detail the production of human serum albumin inplants and U.S. Pat. Nos. 5,202,422, 5,639,947 and 5,959,177 relate tothe production of antibodies in plants. One of the significantadvantages offered by plant-based recombinant protein production systemsis that by increasing the acreage of plants grown, protein productioncan be inexpensively scaled up to provide for large quantities ofprotein. By contrast, fermentation and cell culture systems have largespace, equipment and energy requirements, rendering scale-up ofproduction costly. However, despite the fact that the use of plants asbioreactors is amply documented, and despite the above mentionedtherapeutic applications of apolipoproteins, the prior art provides nomethods for the production of apolipoproteins in plants.

In order to offer a practical alternative to the fermentation and cellculture based systems, it is important that plants remain healthy andthat apolipoproteins accumulate to significant levels in the plants. Inview of the inherent property of apolipoproteins to associate withlipids, recombinantly expressed apolipoproteins may associate with theendogenous plant lipids and thereby interfere with the plant's lipidmetabolism. Thus recombinant expression of apolipoproteins may affectthe health of the plant. Alternatively, the recombinantly expressedapolipoprotein may fail to accumulate to effective levels, as protectivemechanisms may result in degradration of the apolipoprotein. It thus isunclear whether and how the synthetic capacity of plants may beharnessed to achieve the commercial production of apolipoproteins inplants.

Thus in view of the shortcomings associated with the methods for therecombinant production of apolipoproteins by the prior art, there is aneed in the art to improve methods for the production ofapolipoproteins.

SUMMARY OF THE INVENTION

The present invention relates to methods for the production ofapolipoprotein in plants. In particular the present invention relates tomethods for the production of apolipoprotein in plant seeds.

Accordingly, the present invention provides a method for the expressionof an apolipoprotein in plants comprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell; and    -   (c) growing the plant cell into a mature plant capable of        expressing the apolipoprotein.

In accordance with the present invention plant seeds have been found tobe particularly suitable for the production of apolipoprotein.Accordingly, the present invention provides a method for expressingapolipoprotein in plant seeds comprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant seed cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell; and    -   (c) growing the plant cell into a mature plant capable of        setting seed wherein the seed expresses the apolipoprotein.

In a further preferred embodiment the nucleic acid sequence capable ofcontrolling expression in a plant seed cell is a seed-preferredpromoter, such as the phaseolin promoter. In a preferred embodiment, atleast 0.25% of the total seed protein is apolipoprotein.

In preferred embodiments of the present invention the chimeric nucleicacid sequence further comprises a nucleic acid sequence encoding astabilizing polypeptide linked in reading frame to the nucleic acidsequence encoding the apolipoprotein. Preferably the stabilizingpolypeptide is a polypeptide that in the absence of the apolipoproteincan readily be expressed and stably accumulates in a plant cell. Thestabilizing protein may be plant specific or non plant specific.Plant-specific stabilizing polypeptides that can be used in accordancewith the present invention include oil body proteins and thioredoxins.Non-plant specific stabilizing polypeptides that may be used inaccordance herewith include green fluorescent protein (GFP) and singlechain antibodies or fragments thereof. The plant-specific or non-plantspecific stabilizing polypeptide may be linked to the apolipoprotein viaa linker which can be cleaved to release the apolipoprotein in its freenative form.

The chimeric nucleic acid sequence further preferably comprise atargeting signal in such a manner that the apolipoprotein polypeptideaccumulates in the endoplasmic reticulum (ER) or in association with anER-derived storage vesicle, for example an oil body, within the plantcell. Accordingly, the chimeric nucleic acid construct additionally maycomprise a nucleic acid sequence encoding a polypeptide which is capableof targeting the apolipoprotein polypeptide to the ER or an ER derivedstorage vesicle. Nucleic acid sequences that may be used to target theapolipoprotein to the ER include for example nucleic acid sequencesencoding KDEL, HDEL, SDEL sequences. Nucleic acid sequences that may beused to target the apolipoprotein to an oil body include nucleic acidsequences encoding oil body proteins, such as oleosins. In addition, inaccordance with the present invention, the apolipoprotein may betargeted to the oil body by expressing the apolipoprotein in such amanner that the apolipoprotein does not include a targeting signal,provided however, that the nucleic acid sequence encoding theapolipoprotein comprises an apolipoprotein pro-peptide.

In another preferred embodiment the chimeric nucleic acid comprises atargeting signal in such a manner that the apolipoprotein accumulates inthe apoplast. Accordingly, in such an embodiment the chimeric nucleicacid construct additional preferably contains a nucleic acid sequenceencoding a polypeptide which is capable of targeting the apolipoproteinpolypeptide to the apoplast.

In yet a further preferred embodiment, the nucleic acid sequenceencoding the apolipoprotein is expressed in such a manner that theapolipoprotein accumulates in the cytoplasm. In such an embodiment thenucleic acid sequence does not comprise a targeting signal.

In a further preferred embodiment the chimeric nucleic acid construct isintroduced into the plant cell under nuclear genomic integrationconditions. Under such conditions the chimeric nucleic acid sequence isstably integrated in the plant's genome.

In a yet further preferred embodiment the nucleic acid sequence encodingapolipoprotein is optimized for plant codon usage. Preferred nucleicacid sequences used in accordance with the present invention encodehuman, bovine or porcine Apolipoprotein A-I and pro-Apolipoprotein A-I.

In another aspect, the present invention provides a method of obtainingplant seed comprising apolipoprotein. Accordingly, pursuant to thepresent invention a method is provided for obtaining plant seedcomprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant tissue cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell;    -   (c) growing the plant cell into a mature plant capable of        setting seed; and    -   (d) obtaining seed from said plant wherein the seed comprises        the apolipoprotein.

The seeds may be used to obtain a population of progeny plants eachcomprising a plurality of seeds expressing apolipoprotein. The presentinvention also provides plants capable of setting seed expressingapolipoprotein. In a preferred embodiment of the invention, the plantscapable of setting seed comprise a chimeric nucleic acid sequencecomprising in the 5′ to 3′ direction of transcription:

-   -   (a) a first nucleic acid sequence capable of controlling        expression in a plant cell operatively linked to;    -   (b) a second nucleic acid sequence encoding an apolipoprotein        polypeptide, wherein the cell contains the apolipoprotein.

In a preferred embodiment the chimeric nucleic acid sequence isintegrated in the plant's nuclear genome.

In a further preferred embodiment of the present invention the plantthat is used is an Arabidopsis plant and in a particularly preferredembodiment the plant is a safflower plant.

In yet another aspect, the present invention provides plant seedsexpressing apolipoprotein. In a preferred embodiment of the presentinvention, the plant seeds comprise a chimeric nucleic acid sequencecomprising in the 5′ to 3′ direction of transcription:

-   -   (a) a first nucleic acid sequence capable of controlling        expression in a plant cell operatively linked to;    -   (b) a second nucleic acid sequence encoding an apolipoprotein        polypeptide.

The seeds are a source whence the desired apolipoprotein polypeptide,which is synthesized by the seed cells, may be extracted and obtained ina more or less pure form. The apolipoprotein may be used to treatvascular diseases.

Other features and advantages of the present invention will becomereadily apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomereadily apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1. (A) Nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of Homo sapiens Apolipoprotein A-I (Apo AI)(Kindly provided by Dr. Norman Wong, Calgary Alberta) (Accession numberNM_(—)000039). Bold residues represent pre-sequence signal peptide,underlined residues indicate pro-sequence. (B) Nucleotide sequence (SEQID NO:3) and deduced amino acid sequence (SEQ ID NO:4) for naturalvariant Apo AI_(Milano) (R173C). Bold/Italicized residues represents themutated amino acid. (C) Nucleotide sequence (SEQ ID NO:5) and deducedamino acid sequence (SEQ ID NO:6) for natural variant Apo AI Paris(R151C). Bold/Italicized residues represents the mutated amino acid.

FIG. 2. Schematic drawing of all the binary constructs created forapolipoprotein Apo AI expression in Arabidopsis transgenic plants. (A)Constructs that are targeted to the cytosol in the plant cell. (B)Constructs that are targeted to oil bodies in the plant cell. (C-E)Constructs that are targeted to the secretory pathway. (D & E)Constructs may contain additional KDEL sequences to be retained in theendoplasmic reticulum. (D) Constructs containing the pro-sequence of ApoAI or mature Apo AI. (E) Constructs containing a cleavable sequence forthe release of pro-Apo AI or mature Apo AI. Legend describes the type ofpromoter, signal peptide and coding sequence contained in eachconstruct.

FIG. 3(A). Schematic drawing for the cloning strategy for the GFP codingregion to be used in the Apo AI-GFP translational fusion constructs.

FIG. 3(B). Schematic drawing for the cloning strategy for the pro- andmature coding regions of Apo AI and seed-specific Apo AI-GFPtranslational fusion constructs.

FIG. 3(C). Schematic drawing for the cloning strategy for creation ofthe binary vectors used to transform Agrobacterium EHA101 cells forseed-specific cytosolic and oil-body based targeting of Apo AI-GFP.Apo10 and Apo11 containing the mature and pro-Apo AI-GFP, respectively,targeted to the cytosol. Apo12 and Apo13 containing the pro- and matureApo AI-GFP translational fusion, respectively, targeted to oil bodies.

FIG. 3(D). Schematic drawing for the cloning strategy for creation ofthe binary vectors used to transform Agrobacterium EHA101 cells forseed-specific secretory pathway targeting of Apo AI-GFP. Apo15 and Apo16containing the mature and pro-Apo AI-GFP translational fusion,respectively, targeted to the secretory pathway.

FIG. 4(A). Schematic drawing for the cloning strategy for creation ofthe binary vectors used to transform Agrobacterium EHA101 cells forconstitutive cytosolic targeting of Apo AI-GFP. Apo17 and Apo18acontaining the mature and pro-Apo AI-GFP translational fusion,respectively, targeted to the cytosol.

FIG. 4(B). Schematic drawing for the cloning strategy for constitutivecytosolic targeting of Apo AI-GFP. Apo18b containing the pro-Apo AI-GFPtranslational fusion targeted to the cytosol.

FIG. 5(A). Schematic drawing for the cloning strategy for the pro- andmature coding regions of Apo AI and constitutive expression of ApoAI-GFP translational fusion protein.

FIG. 5(B). Schematic drawing for the cloning strategy for the creationof binary vectors used to transform Agrobacterium EHA101 cells forconstitutive secretory pathway targeting of Apo AI-GFP. Apo19 and Apo20containing the mature and pro-Apo AI-GFP translational fusion,respectively, targeted to the secretory pathway.

FIG. 6. Schematic drawing for the cloning strategy for the mature formof the coding region of Apo AI. Apo21 for seed-specific targeting to thecytosol, Apo23 for seed-specific targeting to oil bodies, Apo25 forseed-specific targeting to oil-bodies and purification with cleavagesequence klip8 and Apo29 for seed-specific targeting to the secretorypathway.

FIG. 7. Schematic drawing for the cloning strategy for the pro-form ofthe coding region of Apo AI. Apo22 for seed-specific targeting to thecytosol, Apo24 for seed-specific targeting to oil bodies, Apo30 forseed-specific targeting to the secretory pathway and Apo26 forseed-specific targeting to oil-bodies and purification with cleavagesequence klip8.

FIG. 8(A). Schematic drawing for the cloning strategy for the pro-formof the coding region of Apo AI with the internal XhoI sites removed andcontains a KDEL signal peptide. Apo27 for seed-specific targeting to theoil bodies as an in-frame fusion with oleosin. Apo31 and Apo35 bothtargeted to the secretory pathway, with Apo31 and Apo35 fused in-framewith the oleosin antibody D9, and Apo35 accumulating in the endoplasmicreticulum due to a KDEL signal peptide.

FIG. 8(B). Schematic drawing for the cloning strategy for the pro-formof the coding region of Apo AI_(Milano). Apo27M for seed-specifictargeting of Apo AI_(Milano) to the oil bodies as an in-frame fusionwith oleosin.

FIG. 9. Schematic drawing for the cloning strategy for the pro-form ofthe coding region of Apo AI with the internal XhoI sites removed. Apo28,targeted to oil bodies and able to be cleaved at the klip8 sequence,Apo32 which targets Apo AI to the secretory pathway fused in-frame tothe oleosin antibody D9.

FIG. 10. Schematic drawing for the cloning strategy for the pro- andmature forms of the coding region of Apo AI with the internal XhoI sitesremoved containing an additional Met residue at start of coding region.Apo34 (pro-Apo AI) and Apo33 (mature Apo AI) which are targeted to thesecretory pathway and are fused in-frame with the oleosin antibody D9.

FIG. 11. Schematic drawing for the cloning strategy for the pro-form ofthe coding region of Apo AI with the internal XhoI sites containing aKDEL signal peptide. Apo36, targeted to the secretory pathway and isfused in-frame with the oleosin antibody D9, and accumulates in theendoplasmic reticulum due to a KDEL signal peptide.

FIG. 12. Schematic drawing for the cloning strategy for the pro- andmature forms of the coding region of Apo AI with the internal XhoI sitesremoved containing an additional Met residue at start of coding regionand a KDEL signal peptide. Apo38 and Apo37 which are targeted to thesecretory pathway and are fused in-frame with the oleosin antibody D9,and accumulate in the endoplasmic reticulum due to a KDEL signalpeptide.

FIG. 13. Schematic drawing for the cloning strategy for the pro- andmature forms of the coding region of Apo AI and a protease cleavagesite. Apo43, Apo44, Apo39 and Apo40 targeted to the secretory pathway,fused in-frame with the oleosin antibody D9. Apo43 and Apo44 wouldaccumulate in the endoplasmic reticulum due to a KDEL signal peptide.

FIG. 14. Schematic drawing for the cloning strategy for the pro- andmature forms of the coding region of Apo AI, containing an additionalMet residue at start of coding region and a protease cleavage site.Apo42, Apo46, Apo41, and Apo45 targeted to the secretory pathway, fusedin-frame with the oleosin antibody D9. Apo46 and Apo45 would accumulatein the endoplasmic reticulum due to a KDEL signal peptide.

FIG. 15. Schematic drawing for the cloning strategy for the mature formof the coding region of Apo AI, containing an additional Met residue atstart of coding region and a protease cleavage site. Apo47 is fusedin-frame with the maize oleosin for targeting to oil bodies.

FIG. 16. Westerns of total leaf protein (A) (25 ug) and total seedprotein (B) (50 ug) with polyclonal Apo AI antibody. Apo17 isubi-mat-Apo AI-GFP construct. c24 leaf (25 ug) and seed protein (50 ug)were used as a negative control and 0.5 ug of human blood serum proteinwas used as a positive control.

FIG. 17. Westerns of total leaf protein (A) (25 ug) and total seedprotein (B) (50 ug) with polyclonal Apo AI antibody. Apo18a isubi-pro-Apo AI-GFP construct. c24 leaf (25 ug) and seed protein (50 ug)were used as a negative control and 0.5 ug of human blood serum proteinwas used as a positive control.

FIG. 18. Westerns of total leaf protein (A) (25 ug) and total seedprotein (B) (50 ug) with polyclonal Apo AI antibody. Apo19 isubi-PRS-mat-Apo AI-GFP construct. c24 leaf (25 ug) and seed protein (50ug) were used as a negative control and 0.5 ug of human blood serumprotein was used as a positive control.

FIG. 19. Westerns of total leaf protein (A) (25 ug) and total seedprotein (B) (50 ug) with polyclonal Apo AI antibody. Apo20 isubi-PRS-pro-Apo AI-GFP construct. c24 leaf (25 ug) and seed protein (50ug) were used as a negative control and 0.5 ug of human blood serumprotein was used as a positive control.

FIG. 20. Westerns of total seed protein (50 ug) with polyclonal GFPantibody. (A) Apo10 is pha-mat-Apo AI-GFP construct. (B) Apo11 ispha-pro-Apo AI-GFP construct. c24 seed protein (50 ug) was used as anegative control and 200 ng of purified GFP protein was used as apositive control.

FIG. 21. Westerns of total seed protein (50 ug) with polyclonal GFPantibody. (A) Apo12 is pha-oleosin-mat-Apo AI-GFP construct. (B) Apo13is pha-oleosin-pro-Apo AI-GFP construct. c24 seed protein (50 ug) wasused as a negative control and 200 ng of purified GFP protein was usedas a positive control.

FIG. 22. Westerns of total seed protein (50 ug) with polyclonal GFPantibody. (A) Apo15 is pha-PRS-mat-Apo AI-GFP construct. (B) Apo16 ispha-PRS-pro-Apo AI-GFP construct. c24 seed protein (50 ug) was used as anegative control and 200 ng of purified GFP protein was used as apositive control.

FIG. 23. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo21 is pha-matApo AI. (B) Apo22 is pha-pro-Apo AI. c24seed protein (50 ug) was used as a negative control and 0.5 ug of humanblood serum protein was used as a positive control.

FIG. 24. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo23 is pha-oleo-matApo AI. (B) Apo24 is pha-oleo-pro-ApoAI. c24 seed protein (50 ug) was used as a negative control and 0.5 ugof human blood serum protein was used as a positive control.

FIG. 25. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo25 is pha-oleo-klip8-matApo AI(+Met). (B) Apo26 ispha-oleo-klip8-pro-Apo AI(+Met). c24 seed protein (50 ug) was used as anegative control and 0.5 ug of human blood serum protein was used as apositive control.

FIG. 26. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo28 is pha-oleo-klip8-pro-Apo AI. (B) Apo29 ispha-PRS-mat-Apo AI. c24 seed protein (50 ug) was used as a negativecontrol and 0.5 ug of human blood serum protein was used as a positivecontrol.

FIG. 27. Western of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo30 is pha-PRS-pro-Apo AI. c24 seed protein (50 ug) wasused as a negative control and 0.5 ug of human blood serum protein wasused as a positive control.

FIG. 28. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo32 is pha-PRS-D9 scFv-pro-Apo AI. (B) Apo33 ispha-PRS-D9 scFv-mat-Apo AI(+met). c24 seed protein (50 ug) was used as anegative control and 0.5 ug of human blood serum protein was used as apositive control.

FIG. 29. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo34 is pha-PRS-D9 scFv-pro-Apo AI(+met). (B) Apo35 ispha-PRS-D9 scFv-mat-Apo AI-KDEL. c24 seed protein (50 ug) was used as anegative control and 0.5 ug of human blood serum protein was used as apositive control.

FIG. 30. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo36 is pha-PRS-D9 scFv-pro-Apo AI-KDEL. (B) Apo37 ispha-PRS-D9 scFv-mat-Apo AI(+met)-KDEL. c24 seed protein (50 ug) was usedas a negative control and 0.5 ug of human blood serum protein was usedas a positive control.

FIG. 31. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo38 is pha-PRS-D9 scFv-pro-Apo AI(+met)-KDEL. c24 seedprotein (50 ug) was used as a negative control and 0.5 ug of human bloodserum protein was used as a positive control. (B) Apo 29 is pha-PRS-D9scFv-KLIP8-Apo AI. Wild type seed was used as a negative control and 3ug of human Apo AI from normal human plasma (US Biologicals) was used asa positive control.

FIG. 32. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo40 is pha-PRS-D9 scFv-klip8-pro-Apo AI. (B) Apo41 ispha-PRS-D9 scFv-klip8-mat-Apo AI(+met). c24 seed protein (50 ug) wasused as a negative control and 0.5 ug of human blood serum protein wasused as a positive control.

FIG. 33. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo42 is pha-PRS-D9 scFv-klip8-pro-Apo AI(+met). c24 seedprotein (50 ug) was used as a negative control and 0.5 ug of human bloodserum protein was used as a positive control.

FIG. 34. Westerns of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo44 is pha-PRS-D9 scFv-klip8-pro-Apo AI-KDEL. (B) Apo45is pha-PRS-D9 scFv-klip8-mat-Apo AI(+met). c24 seed protein (50 ug) wasused as a negative control and 0.5 ug of human blood serum protein wasused as a positive control.

FIG. 35. Western of total seed protein (50 ug) with polyclonal Apo AIantibody. (A) Apo46 is pha-PRS-D9 scFv-klip8-pro-Apo AI(+met)-KDEL. c24seed protein (50 ug) was used as a negative control and 0.5 ug of humanblood serum protein was used as a positive control.

FIG. 36. Examination of untargeted and oil body targeted Apo AI-GFPassociation with specific cellular fractions from seeds. Western blotanalysis using the polyclonal Apo AI antibody and approximately equalquantities of total protein (50 μg) isolated from the aqueous (AQ)fraction, phosphate (PW) and urea (UW) washes of oil bodies (PO and UOrespectively, with approximately 20 μg of total oil bodies used) and themicrosomal (ER) fraction from mature seeds. Ponceau-S staining of theimmunoblot shows relative protein amounts loaded on the gel (upperpanel). Human blood serum (0.5 μg) was used as a positive control forApo AI expression. (A) Apo10 is Apo AI-GFP. Apo11 is pro-Apo AI-GFP. (B)Apo12 is oleosin-Apo AI-GFP and Apo13 is oleosin-pro-Apo AI-GFP.

FIG. 37. Examination of secreted pro- and mature Apo AI-GFP associationwith specific cellular fractions from seeds. Western blot analysis usingthe anti-Apo AI antibody and approximately equal quantities of totalprotein (50 _g) isolated from the aqueous (AQ) fraction, phosphate (PW)and urea (UW) washes of oil bodies (PO and UO respectively, withapproximately 20 μg of total oil body protein used) and the microsomal(ER) fraction from mature seeds. Ponceau-S staining of the immunoblotshows relative protein amounts loaded on the gel (upper panel). Humanblood serum (0.5 μg) was used as a positive control for Apo AIexpression. Apo15 is PRS-Apo AI-GFP. Apo16 is PRS-pro-Apo AI-GFP.

FIG. 38. Constitutive expression of Apo AI-GFP translational fusionconstructs in leaves. Western blot analysis using the anti-GFP antibodyand approximately equal quantities of total protein (50 rig) isolatedfrom leaves. Ponceau-S staining of the immunoblot shows relative proteinamounts loaded on the gel (upper panel). Wild-type (c24) plants wereused as a negative control for GFP expression. Purified GFP protein (200ng) was used as a positive control for GFP. UG-14 and UR2 are includedas positive controls for GFP protein accumulation in leaves. Theexpected masses are as follows: Apo17=55.4 kDa; Apo18=56.4 kDa;Apo19=58.3 kDa; Apo20=59.3 kDa; UG-14=26.8 kDa; UR2=50 kDa.

FIG. 39. Seed-specific expression of untargeted and secreted Apo AI inseeds. Western blot analysis using the anti-Apo AI antibody andapproximately equal quantities of total protein (50 μg) isolated frommature seeds. Ponceau-S staining of the immunoblot shows relativeprotein amounts loaded on the gel (upper panel). Wild-type (c24) plantswere used as a negative control for Apo AI expression. Human blood serum(0.5 μg) was used as a positive control for Apo AI expression. Theexpected masses are as follows: Apo21=28.3 kDa; Apo22=29.32 kDa;Apo23=46.9 kDa; Apo24=47.8 kDa; Apo25=51.5 kDa; Apo26=52.5 kDa;Apo27=51.3 kDa; Apo28=52.3 kDa; Apo29=31.3 kDa; Apo30=32.2 kDa.

FIG. 40. Examination of subcellular fractions of Apo22-3 (untargetedpro-Apo AI) T3 generation seeds. Western blot analysis using theanti-Apo AI antibody and approximately equal quantities of total protein(50 μg) isolated from the aqueous (AQ) fraction, phosphate (PW) and urea(UW) washes of oil bodies (PO and UO respectively, with approximately 20μg of total oil bodies used) from mature seeds. Ponceau-S staining ofthe immunoblot shows relative protein amounts loaded on the gel (upperpanel). Human blood serum (0.5 μg) was used as a positive control forApo AI expression. Molecular weight sizes are indicated on the left.

FIG. 41. Confocal micrographs of Apo AI-GFP seed-specific constructsexpressed in late cotyledonary stage embyro cells stained with Nile Red.(A-C) Apo10 is untargeted mature Apo AI fused to GFP. (D-F) Apo11 isuntargeted pro-Apo AI fused to GFP. (G-I) Apo12 is mature Apo AI fusedto GFP targeted to oil bodies using oleosin. (Column 1) Green channel.(Column 2) Red channel. (Column 3) Merged channels. Bar=5 μm.

FIG. 42. Confocal micrographs of Apo AI-GFP seed-specific constructsexpressed in late cotyledonary stage embryo cells stained with Nile Red.(A-C) Apo13 is pro-Apo AI fused to GFP targeted to oil bodies usingoleosin. (D-F) Apo15 is mature Apo AI fused to GFP targeted to thesecretory pathway. (G-I) Apo16 is pro-Apo AI fused to GFP targeted tothe secretory pathway. (Column 1) Green channel. (Column 2) Red channel.(Column 3) Merged channels. Bar=5 μm.

FIG. 43. Confocal micrographs of constitutively expressed untargeted andsecreted Apo AI-GFP fusion protein in late cotyledonary stage embryocells stained with Nile Red. (A-C) Apo17 is untargeted mature Apo AIfused to GFP. (D-F) Apo18 is untargeted pro-Apo AI fused to GFP. (G-H)Apo19 is mature Apo AI fused to GFP targeted to the secretory pathway.(J-L) Apo20 is pro-Apo AI fused to GFP targeted to the secretorypathway. (Column 1) Green channel. (Column 2) Red channel. (Column 3)Merged channels. Bar=5 μm.

FIG. 44. Confocal micrographs of Apo AI-GFP constitutive constructsexpressed in leaf epidermal cells. (A-C) Apo17 is untargeted mature ApoAI fused to GFP. (D-F) Apo18 is untargeted pro-Apo AI fused to GFP.(G-H) Apo19 is mature Apo AI fused to GFP targeted to the secretorypathway. (J-L) Apo20 is pro-Apo AI fused to GFP targeted to thesecretory pathway. (Column 1) Green channel. (Column 2) Red channel.(Column 3) Merged channels. Bar=5 μm.

FIG. 45. Purification of Apo25, Apo26 and Apo28 by reverse phasechromatography. (A) HPLC trace of ApoAI standard (B) HPLC trace of Apo25(C) HPLC trace of Apo26 (D) HPLC trace of Apo28. Individual wavelengthsused included 214, 254, 280 and 326 nm.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present invention relates to methods forthe production of apolipoprotein in transgenic plants. The presentinventors have surprisingly found that production of apolipoproteins inplants is not only feasible but also offers substantial advantages overthe conventional methodologies. The raw materials for plant basedproduction are more stable, particularly as the protein is produced inplant seeds, and, moreover, are free of bacterial endotoxins. Thus thepresent invention provides a safe source material for the manufacture ofapolipoproteins. It has also been discovered that recombinant expressionof apolipoprotein can yield the native apolipoprotein in more or lesspure form at levels that permits commercial scale manufacture ofapolipoproteins. Accordingly, pursuant to the present invention a methodfor the expression of a nucleic acid sequence encoding apolipoprotein inplants is provided in which the method comprises:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide    -   (b) introducing the chimeric nucleic acid construct into a plant        cell; and    -   (c) growing the plant cell into a mature plant expressing        apolipoprotein.

The present inventors have found that high levels of apolipoproteinexpression may be achieved by expressing the recombinant protein inplant seeds. Accordingly, the present invention provides a method forexpressing apolipoprotein in plant seeds comprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant seed cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell; and    -   (c) growing the plant cell into a mature plant capable of        setting seed wherein the seed expresses the apolipoprotein.

TERMS AND DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinshall have the same meaning as is commonly understood by one skilled inthe art to which the present invention belongs. Where permitted, allpatents, applications, published applications, and other publications,including nucleic acid and polypeptide sequences from GenBank, SwissProand other databases referred to in the disclosure are incorporated byreference in their entirety.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentinvention may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The terms “nucleic acid sequence encoding apolipoprotein” and “nucleicacid sequence encoding an apolipoprotein polypeptide”, which may be usedinterchangeably herein, refer to any and all nucleic acid sequencesencoding an apolipoprotein polypeptide, including any mammalianapolipoprotein polypeptide and any nucleic acid sequences that encodepro-apolipoprotein and pre-pro-apolipoprotein. As used herein“pro-apolipoprotein” refers to an apolipoprotein polypeptide whichincludes a polypeptide which is cleaved post-translationally. In nativehuman apolipoprotein the pro-peptide is a 6 amino acid residuepolypeptide chain. The term “pre-pro-apolipoprotein” refers to apro-apolipoprotein molecule additionally comprising an N-terminal signalsequence which facilitates intracellular transport of the polypeptidechain. Nucleic acid sequences encoding an apolipoprotein polypeptidefurther include any and all nucleic acid sequences which (i) encodepolypeptides that are substantially identical to the apolipoproteinpolypeptide sequences set forth herein; or (ii) hybridize to anyapolipoprotein nucleic acid sequences set forth herein under at leastmoderately stringent hybridization conditions or which would hybridizethereto under at least moderately stringent conditions but for the useof synonymous codons.

By the term “substantially identical” it is meant that two polypeptidesequences preferably are at least 70% identical, and more preferably areat least 85% identical and most preferably at least 95% identical, forexample 96%, 97%, 98% or 99% identical. In order to determine thepercentage of identity between two polypeptide sequences the amino acidsequences of such two sequences are aligned, using for example thealignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443),as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) sothat the highest order match is obtained between the two sequences andthe number of identical amino acids is determined between the twosequences. Methods to calculate the percentage identity between twoamino acid sequences are generally art recognized and include, forexample, those described by Carillo and Lipton (SIAM J. Applied Math.,1988, 48:1073) and those described in Computational Molecular Biology,Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing:Informatics and Genomics Projects. Generally, computer programs will beemployed for such calculations. Computer programs that may be used inthis regard include, but are not limited to, GCG (Devereux et al.,Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul etal., J. Molec. Biol., 1990:215:403). A particularly preferred method fordetermining the percentage identity between two polypeptides involvesthe Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J,1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci.USA 89: 10915-10919 using a gap opening penalty of 10 and a gapextension penalty of 0.1, so that the highest order match obtainedbetween two sequences wherein at least 50% of the total length of one ofthe two sequences is involved in the alignment.

By “At least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the T_(m), which in sodium containing buffers is afunction of the sodium ion concentration and temperature (T_(m)=81.5°C.−16.6 (Log₁₀ [Na⁺])+0.41(% (G+C)−600/l), or similar equation).Accordingly, the parameters in the wash conditions that determine hybridstability are sodium ion concentration and temperature. In order toidentify molecules that are similar, but not identical, to a knownnucleic acid molecule a 1% mismatch may be assumed to result in about a1° C. decrease in T_(m), for example if nucleic acid molecules aresought that have a >95% identity, the final wash temperature will bereduced by about 5° C. Based on these considerations those skilled inthe art will be able to readily select appropriate hybridizationconditions. In preferred embodiments, stringent hybridization conditionsare selected. By way of example the following conditions may be employedto achieve stringent hybridization: hybridization at 5× sodiumchloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at T_(m)(based on the above equation)−5° C., followed by a wash of 0.2×SSC/0.1%SDS at 60° C. Moderately stringent hybridization conditions include awashing step in 3×SSC at 42° C. It is understood however that equivalentstringencies may be achieved using alternative buffers, salts andtemperatures. Additional guidance regarding hybridization conditions maybe found in: Current Protocols in Molecular Biology, John Wiley & Sons,N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

As used herein the terms “apolipoprotein” and “apolipoproteinpolypeptide” refer to any and all polypeptide sequences of anapolipoprotein including all mammalian apolipoprotein polypeptides and apolypeptide comprising a sequence of amino acid residues which is (i)substantially identical to the amino acid sequences constituting anyapolipoprotein polypeptides set forth herein or (ii) encoded by anucleic acid sequence capable of hybridizing under at least moderatelystringent conditions to any nucleic acid sequence encodingapolipoprotein set forth herein or capable of hybridizing under at leastmoderately stringent conditions to any nucleic acid sequence encodingapolipoprotein set forth herein but for the use of synonymous codons.The terms apolipoprotein and apolipoprotein polypeptide includepro-apolipoprotein polypeptides. The apolipoprotein polypeptide ispreferably of human, porcine or bovine origin. In a preferredembodiments these apolipoproteins include, but are not limited to,Apolipoprotein A-I (Apo AI), Apolipoprotein A-IV (Apo AIV),Apolipoprotein A-V (Apo AV) and Apolipoprotein E (Apo E).

The term “chimeric” as used herein in the context of nucleic acidsequences refers to at least two linked nucleic acid sequences which arenot naturally linked. Chimeric nucleic acid sequences include linkednucleic acid sequences of different natural origins. For example anucleic acid sequence constituting a plant promoter linked to a nucleicacid sequence encoding human apolipoprotein is considered chimeric.Chimeric nucleic acid sequences also may comprise nucleic acid sequencesof the same natural origin, provided they are not naturally linked. Forexample a nucleic acid sequence constituting a promoter obtained from aparticular cell-type may be linked to a nucleic acid sequence encoding apolypeptide obtained from that same cell-type, but not normally linkedto the nucleic acid sequence constituting the promoter. Chimeric nucleicacid sequences also include nucleic acid sequences comprising anynaturally occurring nucleic acid sequence linked to any non-naturallyoccurring nucleic acid sequence.

Preparation of Recombinant Expression Vectors Comprising ChimericNucleic Acid Sequences Encoding Apolipoprotein and a Nucleic AcidSequence Capable of Controlling Expression in a Plant Cell

The nucleic acid sequences encoding apolipoprotein that may be used inaccordance with the methods and compositions provided herein may be anynucleic acid sequence encoding an apolipoprotein polypeptide, includingany proapolipoprotein and preproapolipoprotein.

There are a number of different apolipoproteins that are found in humanblood plasma, and they can act as signals, that cause lipoproteins toact on certain tissues or that activate enzymes that act on thoselipoproteins (Lehninger, A. et al. Principles of Biochemistry, secondedition, New York, Worth Publishers, 1993). These proteins include butare not limited to alleles and isoforms of apolipoprotein A-I (Apo AI)(see for example Sharpe C R et al., Nucleic Acids Res. 12 (9), 3917-3932(1984)), Apo AII ((see for example Sharpe C R et al., Nucleic Acids Res.12 (9), 3917-3932 (1984)), Apo AIV (see for example Elshourbagy N A etal, J. Biol. Chem. 261 (5), 1998-2002 (1986)), Apo AV (see for exampleHubacek et al. Physiol. Res. 2004. 53:225-228), Apo B-100 (see forexample Law S W et al., Proc. Natl. Acad. Sci. U.S.A. 83 (21), 8142-8146(1986)), Apo B-48 (see for example Powell L M et al., Cell 50 (6),831-840 (1987)), Apo C-II (see for example Sharpe C R et al., NucleicAcids Res. 12 (9), 3917-3932 (1984)), Apo C-III (see for example SharpeC R et al., Nucleic Acids Res. 12 (9), 3917-3932 (1984)), ApoC-IV (seefor example Allan C M et al., Genomics 28 (2), 291-300 (1995)), Apo D(Drayna D et al. J. Biol. Chem. 261 (35), 16535-16539 (1986)), Apo E(see for example Brewslow J L et al., J. Biol. Chem. 257 (24),14639-14641 (1982)), Apo F (see for example Day J R et al., Biochem.Biophys. Res. Commun. 203 (2), 1146-1151 (1994)), Apo H (see for exampleMehdi, H., et al., Gene 108 (2), 293-298 (1991) and Apo L (see forexample Duchateau, P. N., et al., J. Biol. Chem. 272 (41), 25576-25582(1997)). Exemplary nucleic acid sequences encoding apolipoprotein arewell known to the art and are generally readily available from a diversevariety of mammalian sources including human (see above), porcine (seefor example Trieu V N et al., Gene 134 (2), 267-270 (1993)), bovine (seefor example Yang, Y. W., et al. J. Mol. Evol. 32 (6), 469-475 (1991)),ovine (see for example Robertson, J. A., et al., J. Steroid Biochem.Mol. Biol. 67 (4), 285-292 (1998)) and the like. Human apolipoproteinencoding sequences that may be used include those encoding polypeptidechains set forth as SEQ ID NO:1, 7 and 8. Further non-humanapolipoprotein encoding sequences that may be used in accordance of thepresent invention are set forth in SEQ ID NO: 9-55 and 241-251. Therespective corresponding nucleic acid sequences encoding theapolipoprotein polypeptide chains can be readily identified via theAccession identifier numbers provided in Table 1. Using these nucleicacid sequences, additional novel apolipoprotein encoding nucleic acidsequences may be readily identified using techniques known to those ofskill in the art. For example libraries, such as expression libraries,cDNA and genomic libraries, may be screened, and databases containingsequence information from sequencing projects may be searched forsimilar sequences. Alternative methods to isolate additional nucleicacid sequences encoding apolipoprotein polypeptides may be used, andnovel sequences may be discovered and used in accordance with thepresent invention. In preferred embodiments nucleic acid sequencesencoding apolipoprotein are human, porcine and bovine apolipoprotein.

Numerous apolipoprotein analogs are known to the prior art (see forexample Cheung M C et al., Biochim Biophys Acta. 1988 May 2;960(1):73-82 and Strobl W et al., Pediatr Res. 1988 August; 24(2):222-8)and may be used in accordance with the present invention. Analogs thatmay be used herein include human apolipoprotein molecules wherein avariety of natural and synthetic mutations and modifications have beendiscovered including, but not limited to, point mutations, deletionmutations, frameshift mutations and chemical modifications. Inaccordance with the present invention in a preferred embodiment, thenatural variant known as Apo AI-M is used. Examples of mutations andmodifications that may be used in accordance with the present inventioninclude, but are not limited to, those set forth in Table 2.

In preferred embodiments, the nucleic acid sequence encodingapolipoprotein that is used is pro-apolipoprotein.

Alterations to the nucleic acid sequence encoding apolipoprotein toprepare apolipoprotein analogs may be made using a variety of nucleicacid modification techniques known to those skilled in the art,including for example site directed mutagenesis, targeted mutagenesis,random mutagenesis, the addition of organic solvents, gene shuffling ora combination of these and other techniques known to those of skill inthe art (Shraishi et al., 1988, Arch. Biochem. Biophys, 358: 104-115;Galkin et al., 1997, Protein Eng. 10: 687-690; Carugo et al., 1997,Proteins 28: 10-28; Hurley et al., 1996, Biochem, 35 : 5670-5678;Holmberg et al., 1999, Protein Eng. 12 : 851-856).

In accordance herewith the nucleic acid sequence encoding apolipoproteinis linked to a nucleic acid sequence capable of controlling expressionof the apolipoprotein polypeptide in a plant cell. Accordingly, thepresent invention also provides a nucleic acid sequence encodingapolipoprotein linked to a promoter capable of controlling expression ina plant cell. Nucleic acid sequences capable of controlling expressionin plant cells that may be used herein include any plant derivedpromoter capable of controlling expression of polypeptides in plants.Generally, promoters obtained from dicotyledonous plant species will beused when a dicotyledonous plant is selected in accordance herewith,while a monocotyledonous plant promoter will be used when amonocotyledonous plant species is selected. In one embodiment, apromoter is used which results in the expression of the apolipoproteinpolypeptide in the entire plant. Constitutive promoters that may be usedinclude, for example, the 35S cauliflower mosaic virus (CaMV) promoter(Rothstein et al., 1987, Gene 53: 153-161), the rice actin promoter(McElroy et al., 1990, Plant Cell 2:163-171; U.S. Pat. No. 6,429,357), aubiquitin promoter, such as the corn ubiquitin promoter (U.S. Pat. Nos.5,879,903; 5,273,894), and the parsley ubiquitin promoter (Kawalleck, P.et al., 1993, Plant Mol. Biol. 21:673-684).

In particularly preferred embodiments of the present invention, theapolipoprotein is produced in plant seeds. Production in plants seedsoffers flexibility in storage and shipment of apolipoprotein as a rawmaterial, since apolipoprotein retains its activity upon extraction fromstored seed. Furthermore, the amount of biomass that needs to besubjected to extraction is limited due to the relatively low watercontent present in plant seeds. Accordingly, in a preferred embodimentof the present invention the plant cell is a seed cell and the plant isgrown into a mature plant capable of setting seed wherein the seedexpresses the apolipoprotein. In a further preferred embodiment thenucleic acid sequence capable of controlling expression in a plant cellis a seed-preferred promoter, such as the phaseolin promoter. In such anembodiment a promoter which results in preferential expression of theapolipoprotein polypeptide in seed tissue is used. “Seed-preferredpromoters” in this regard are promoters which control expression of arecombinant protein (i.e. apolipoprotein) so that preferably at least80% of the total amount of recombinant protein present in the matureplant is present in the seed. More preferably at least 90% of the totalamount of recombinant protein present in the mature plant is present inthe seed. Most preferably at least 95% of the total amount ofrecombinant protein present in the mature plant is present in the seed.Seed-preferred promoters that may be used in this regard include, forexample, the bean phaseolin promoter (Sengupta-Gopalan et al., 1985,Proc. Natl. Acad. Sci. USA 82: 3320-3324); the Arabidopsis 18 kDaoleosin promoter (U.S. Pat. No. 5,792,922) or the flax oleosin promoter(WO 01/16340); the flax legumin like seed storage protein (linin)promoter (WO 01/16340); the flax 2S storage protein promoter (WO01/16340); an endosperm preferred promoter such as the Amy32b promoter(Rogers and Milliman, J. Biol. Chem., 1984, 259: 12234-12240, the Amy6-4promoter (Kursheed and Rogers, J. Biol. Chem., 1988, 263: 18953-18960 orthe Aleurain promoter (Whittier et al., 1987, Nucleic Acids Res., 15:2515-2535) or the bean arcelin. promoter (Jaeger G D, et al., 2002, Nat.Biotechnol. December; 20:1265-8). New promoters useful in various plantsare constantly discovered. Numerous examples of plant promoters may befound in Ohamuro et al. (Biochem. of Pints., 1989, 15: 1-82).

In preferred embodiments of the present invention the chimeric nucleicacid sequence further comprises a nucleic acid sequence encoding astabilizing polypeptide linked in reading frame to the nucleic acidsequence encoding the apolipoprotein. The stabilizing polypeptide isused to facilitate protein folding and/or enhance the stableaccumulation of the apolipoprotein in plant cells. In addition thestabilizing polypeptide may be used to target the apolipoprotein to adesired location within the plant cell and/or facilitate purification ofthe apolipoprotein. Preferably the stabilizing polypeptide is apolypeptide that in the absence of the apolipoprotein can readily beexpressed and stably accumulates in transgenic plant cells. Thestabilizing polypeptide may be a plant specific or non-plant specificpolypeptide. Plant-specific stabilizing polypeptides that can be used inaccordance with the present invention include oil body proteins (Seebelow) and thioredoxins, for example, the thioredoxin shown in SEQ IDNO:56. Non-plant specific stabilizing polypeptides that may be used inaccordance herewith include green fluorescent protein (GFP) (Davis andVierstra, 1996, Weeds World 3(2):43-48) (SEQ ID NO:57) and single chainantibodies or fragments thereof. Preferably non-plant specificstabilizing polypeptides are codon optimized for optimal expression inplants.

Single chain antibodies or antibodies that are preferably used hereininclude single chain antibodies or fragments thereof that facilitatepurification of the apolipoprotein. In accordance herewith, for example,a single chain antibody or fragment thereof which is capable of specificassociation with an oil body or oil body protein may be used, therebypermitting copurification of the apolipoprotein with the oil bodyfraction which can readily be obtained from plant seeds. Preferably thesingle chain antibody is capable of associating with an oil body proteinobtainable from the seed in which the apolipoprotein is expressed, i.e.in an embodiment of the invention in which Arabidopsis plant cells areused, a single chain antibody or fragment thereof is selected which iscapable of associating with an Arabidopsis oil body protein. In afurther preferred embodiment, the single chain antibody is a singlechain FV antibody capable of specifically associating with the 18 kDaoleosin from Arabidopsis thaliana (D9scFv). In the most preferredembodiment, the single chain antibody is shown in SEQ ID NO:240. Theterm “single chain antibody fragment” (scFv) or “antibody fragment” asused herein means a polypeptide containing a variable light (V_(L))domain linked to a variable heavy (V_(H)) domain by a peptide linker(L), represented by V_(L)-L-V_(H). The order of the V_(L) and V_(H)domains can be reversed to obtain polypeptides represented asV_(H)-L-V_(L). “Domain” is a segment of protein that assumes a discretefunction, such as antigen binding or antigen recognition. The singlechain antibody fragments for use in the present invention can be derivedfrom the light and/or heavy chain variable domains of any antibody.Preferably, the light and heavy chain variable domains are specific forthe same antigen. Most preferably the antigen is an oil body protein.The individual antibody fragments which are joined to form a multivalentsingle chain antibody may be directed against the same antigen or can bedirected against different antigens. Methodologies to create singlechain antibodies are well known to the art. For example single chainantibodies can be created by screening single chain (scFV) phage displaylibraries.

Methodologies to create single chain antibodies from phage displaylibraries are well known to the art. McCarrerty et al. (Nature 348:552-554) demonstrated the use of a phage-display system in whichfragments of antibodies were expressed as a fusion protein with a fdphage vector to allow for the expression of single chain antibodies onthe surface of the phage. The production of a single chain antibodyphage display library can be achieved using for example, the RecombinantPhage Antibody System developed by Amersham Biosciences and CambridgeAntibody Technology. A more detailed protocol is available from AmershamBiosciences which is sold as in 3 parts including a mouse scFV molecule,and expression module and a detection module. Briefly, the protocol forthe production of single chain antibodies is as follows. Messenger RNAcan be obtained from either a mouse hybridoma or mouse spleen cells froma mouse that has been immunized with the antigen of interest. The mousehybridoma represents the most abundant source for the antibody gene tobe cloned as it expressed the heavy and light chain genes for a singleantibody but antibodies can also be cloned using spleen cells from animmunized mouse. The mRNA is converted to cDNA using a reversetranscriptase and random hexamer primers. The use of random hexamerswill result in cDNA molecules that are sufficient in length to clone thevariable regions of the heavy and light chain molecules. After the cDNAmolecules are created, primary PCR reactions are performed to amplifythe heavy and light variable regions separately. Primers are designed toamplify the heavy or light chain variable region by hybridizing toopposite ends of the chain. Once the variable regions are amplified, thePCR reactions are subjected to agarose gel electrophoresis and gelpurified to remove the primers and any extraneous PCR products. Once theheavy and light chain variable regions have been purified they areassembled into a single gene using a linker. The linker region isdesigned to ensure that the correct reading frame is maintained betweenthe heavy and light chain. For example, the variable heavy (V_(H)) andvariable light (V_(L)) chains may be linked using a (Gly₄Ser)₃ linker toobtain a single chain antibody fragment (scFv) of approximately 750 basepairs in length. Once the heavy and light chains are assembled with thelinker a secondary PCR reaction is performed to amplify the assembledscFV DNA fragments. Primers should be designed to introduce restrictionsites to allow for cloning into phagemid expression vectors. For exampleSfi I and Not I sites can be added to the 5′ and 3′ end of these scFvgene for cloning into the pCANTAB 5 E vector (Amersham Biosciences).Once PCR is complete, the DNA fragments should be purified to removeunincorporated primers and dNTPs. This can be achieved using spun-columnpurification. Once the DNA fragments have been purified and quantifiedthe fragments are digested with the appropriate restriction enzymes toallow for cloning into the appropriate expression vector. The DNAfragments are subsequently ligated into an expression vector, forexample pCANTAB 5E (Amersham Biosciences) and introduced into competentE. coli cells. The cells should be grown on appropriate selection mediato ensure that only cells containing the expression vector will grow(i.e. using a specific carbon source and antibiotic selection). Once theE. coli is grown, the phagemid-containing colonies are infected with aM13 helper phage (i.e. KO7—Amersham Biosciences) to yield recombinantphage which display the scFv fragments. The M13 phage will initiatephage replication and complete phage particles will be produced andreleased from the cells, expressing scFv species on their surface. Thephage displaying the correct scFv antibodies are identified by panningusing the specific antigen. To eliminate the non-specific phage, theculture of recombinant phage can be transferred to an antigen-coatedsupport (i.e. a flask or a tube), and washed. Only those phagedisplaying the correct scFv will be bound to the support. A susceptiblestrain of E. coli is subsequently infected with the phage bound to theantigen-coated support. The phage can be enriched by rescuing with thehelper phage and panning against the antigen multiple times or can beplated directly onto a solid medium without further enrichment. The E.coli cells that have been infected with the phage selected against theappropriate antigen are plated and individual colonies are picked.Phage, from the individual colonies, are then assayed using for examplethe ELISA assay (enzyme-linked immunosorbent assay). Phage antibodieswhich are positive using the ELISA assay can then be used to infect E.coli HB2151 cells for the production of soluble recombinant antibodies.Once the appropriate clones are selected the sequence of the scFvantibody gene can be identified and used for the present invention.

The stabilizing protein may be linked to the apolipoprotein via a linkerwhich can be cleaved to release the apolipoprotein in its free nativeform. Linkers that may be included in this regard include peptidesequences recognized by Factor Xa, IgA protease, or entorokinase. In aparticularly preferred embodiment the linker encodes a chymosinpro-sequence which may be cleaved with mature chymosin as set forth inPCT Patent Application WO 98/49326.

In preferred embodiments the chimeric nucleic acid sequence furthercomprises a “targeting signal”. Targeting signal as used herein meansany amino acid sequence capable of directing the apolipoproteinpolypeptide, when expressed, to a desired location within the plantcell. Suitable targeting signals that may be used herein include thosecapable of targeting the apolipoprotein polypeptide to the endoplasmicreticulum or a storage vesicle derived from the endoplasmic reticulum,such as an oil body, and the apoplast.

In order to achieve accumulation of the apolipoprotein in the ER or anER-derived storage vesicle, the polypeptide encoding the polypeptideencoding the apolipoprotein is linked to a targeting signal which causesthe apolipoprotein to be retained in the ER or an ER-derived storagevesicle. In a preferred embodiment of the present invention, thetargeting signal that is capable of retaining the apolipoprotein in theER contains a C-terminal ER-retention motif. Examples of such C-terminalER-retention motifs include KDEL, HDEL, DDEL, ADEL and SDEL sequences.Other examples include HDEF (Lehmann et al., 2001, Plant Physiol.127(2): 436-439), or two arginine residues close to the N-terminuslocated at positions 2 and 3, 3 and 4, or 4 and 5 (Abstract from PlantBiology 2001 Program, ASPB, July 2001, Providence, R.I., USA). Nucleicacid sequences encoding a C-terminal retention motif are preferablylinked to the nucleic acid sequence encoding the apolipoprotein in sucha manner that the polypeptide capable of retaining the apolipoprotein inthe ER is linked to the C-terminal end of the apolipoproteinpolypeptide.

In embodiments of the present invention in which the apolipoprotein isretained in the ER the chimeric nucleic acid sequence additionally maycontain a nucleic sequence which targets the nucleic acid sequence tothe endomembrane system (“signal peptide”). In embodiments of thepresent invention in which the apolipoprotein polypeptide is retained inthe ER using a sequence, such as KDEL, HDEL or SDEL polypeptide, it isparticularly desirable to include a nucleic acid sequence encoding asignal peptide. Exemplary signal peptides that may be used hereininclude the tobacco pathogenesis related protein (PR-S) signal sequence(SEQ. ID. NO:58) (Sijmons et al., 1990, Bio/technology, 8:217-221),lectin signal sequence (Boehn et al., 2000, Transgenic Res,9(6):477-86), signal sequence from the hydroxyproline-rich glycoproteinfrom Phaseolus vulgaris (Yan et al., 1997, Plant Phyiol. 115(3):915-24and Corbin et al., 1987, Mol Cell Biol 7(12):4337-44), potato patatinsignal sequence (Iturriaga, G et al., 1989, Plant Cell 1:381-390 andBevan et al., 1986, Nuc. Acids Res. 41:4625-4638.) and the barley alphaamylase signal sequence (Rasmussen and Johansson, 1992, Plant Mol. Biol.18(2):423-7). Example No. 3 herein shows accumulation of theapolipoprotein in the ER.

In a further preferred embodiment, the apoliprotein polypeptide islinked to a polypeptide that is capable of retaining the apolipoproteinpolypeptide in an ER-derived storage vesicle. In a preferred embodiment,the ER derived storage vesicle is an oil body and the apolipoprotein islinked to an oil body protein. Oil body proteins that may be used inthis regard include any protein that naturally associates with an oilbody (see SEQ ID NOs:59-137 in Table 3). The respective correspondingnucleic acid sequences encoding the oil body protein polypeptide chainscan be readily identified via the Accession identifier numbers providedin Table 3. Using these nucleic acid sequences, additional novel oilbody proteins encoding nucleic acid sequences may be readily identifiedusing techniques known to those of skill in the art. For examplelibraries, such as expression libraries, cDNA and genomic libraries, maybe screened, and databases containing sequence information fromsequencing projects may be searched for similar sequences. Alternativemethods to isolate additional nucleic acid sequences encoding oil bodyprotein polypeptides may be used, and novel sequences may be discoveredand used in accordance with the present invention. Oil body proteinsthat are particularly preferred are oleosins, for example a corn oleosin(Bowman-Vance et al., 1987, J. Biol. Chem. 262: 11275-11279; Qu et al.,1990, J. Biol. Chem. 265: 2238-2243 or Brassica (Lee et al., 1991, PlantPhysiol. 96: 1395-1397), caleosins, see for example Genbank accessionnumber AF067857) and steroleosins (Lin et al., 2002 Plant Physiol.128(4):1200-11). In a further preferred embodiment, the oil body proteinis a plant oleosin and shares sequence similarity with other plantoleosins such as the oleosin isolated from Arabidopsis thaliana (SEQ IDNO:138) or Brassica napus (SEQ ID NO:139). In another embodiment, theoil body protein is a caleosin or calcium binding protein from plant,fungal or other sources and shares sequence homology with plantcaleosins such as the caleosin isolated from Arabidopsis thaliana (SEQID NO:140 and SEQ ID NO:141) In another embodiment the oil body proteinis a steroleosin (SEQ ID NO:142), a sterol binding dehydrogenase (LinL-J et al, (2002) Plant Physiol 128: 1200-1211). This embodiment of thepresent invention is exemplified in Example No. 3.

In addition, in accordance with the present invention, theapolipoprotein may also be targeted to an oil body by expressing theapolipoprotein in such a manner that the apolipoprotein does not includea targeting signal, provided however, that the nucleic acid sequenceencoding the apolipoprotein comprises an apolipoprotein pro-peptide.This embodiment of the present invention is exemplified in Example No.3.

Polypeptides capable of retaining the apolipoprotein in the ER or an ERderived storage vesicle are typically not cleaved and the apolipoproteinmay accumulate in the form of a fusion protein, which is, for example,typically the case when a KDEL retention signal is used to retain thepolypeptide in the ER or when an oil body protein is used to retain thepolypeptide in an oil body.

In another embodiment of the present invention the apolipoproteinpolypeptide is expressed in such a manner the polypeptide accumulates inthe apoplast. In order to achieve such accumulation the chimeric nucleicacid sequence preferable comprises a targeting sequence capable ofdirecting the apoliprotein polypeptide to the ER (“signal peptide”).Exemplary signal peptides that may be used herein include thehereinbefore mentioned tobacco pathogenesis related protein (PR-S)signal sequence (SEQ. ID. NO:58) (Sijmons et al., 1990, Bio/technology,8:217-221), lectin signal sequence (Boehn et al., 2000, Transgenic Res,9(6):477-86), signal sequence from the hydroxyproline-rich glycoproteinfrom Phaseolus vulgaris (Yan et al., 1997, Plant Phyiol. 115(3):915-24and Corbin et al., 1987, Mol Cell Biol 7(12):4337-44), potato patatinsignal sequence (Iturriaga, G et al., 1989, Plant Cell 1:381-390 andBevan et al., 1986, Nuc. Acids Res. 41:4625-4638.) and the barley alphaamylase signal sequence (Rasmussen and Johansson, 1992, Plant Mol. Biol.18(2):423-7). Such targeting signals may in vivo be cleaved off from theapolipoprotein polypeptide, which for example is typically the case whenan apoplast targeting signal, such as the tobacco pathogenesis relatedprotein-S (PR-S) signal sequence (Sijmons et al., 1990, Bio/technology,8:217-221) is used. Other signal peptides can be predicted using theSignalP World Wide Web server (http://www.cbs.dtu.dk/services/SignalP/)which predicts the presence and location of signal peptide cleavagesites in amino acid sequences from different organisms. In general thereis little conservation of the primary amino acid sequence, althoughgeneral physiochemical properties are conserved to some extent. Thegeneric structure of signal peptides has 3 regions, the shortamino-terminal “n-region” contains positively charged residues, thecentral hydrophobic “h-region” ranges in size from 7 to 15 amino acidsand the carboxy-terminal “c-region” contains polar amino acids and acleavage site that is recognized by membrane bound signal peptidaseenzymes (Nakai K., 2000, Advances in Protein Chem 54:277-344). Atargeting signal that also may be used in accordance herewith includesthe native apolipoprotein signal sequence (18 amino acids in length incase of the human sequence). In preferred embodiments hereof anN-terminally located apoplast targeting sequence, such as thehereinbefore mentioned tobacco PR-S sequence is used combined with aC-terminally located ER retention sequence such as the KDEL sequence.

In yet a further preferred embodiment, the nucleic acid sequenceencoding the apolipoprotein is expressed in such a manner that theapolipoprotein accumulates in the cytoplasm. In such an embodiment thenucleic acid sequence does not comprise a targeting signal. Preferablyin such an embodiment the apolipoprotein comprises a further stabilizingpolypeptide, such as green fluorescent protein (GFP).

The chimeric nucleic acid sequence may also comprise N- and/orC-terminal polypeptide extensions. Such extensions may be used tostabilize and/or assist in folding of the apolipoprotein polypeptidechain or they may facilitate targeting to a compartment in the cell, forexample the oil body. Polypeptide extensions that may be used in thisregard include for example a nucleic acid sequence encoding a singlechain antibody, or a nucleic acid sequence encoding green fluorescentprotein (Davis and Vierstra, 1996, Weeds World 3(2):43-48), orcombinations of such polypeptides. Single chain antibody extensions thatare particularly desirable include those that permit association of theapoliprotein with an oil body in order to facilitate purification of theapolipoprotein in association with the oil body fraction. Suchextensions are preferably included in embodiments of the presentinvention in which the apolipoprotein is expressed in the plant seed andtargeted within the seed cell to the ER or to the apoplast.

In a further embodiment, a cleavage site may be located upstream of theN-terminus or downstream of the C-terminus of the Apolipoprotein A-Ipeptide allowing for the Apolipoprotein A-I polypeptide to be cleavedfrom the fusion partner, thereby obtaining isolated Apolipoprotein A-I.Examples of such cleavage sites can be found in WO 98/49326 (Method forthe cleavage of fusion proteins) and related applications and LaVallieet al. (1994) Enzymatic and chemical cleavage of fusion proteins InCurrent Protocols in Molecular Biology pp 16.4.5-16.4.17, John Wiley andSons, Inc., New York N.Y. In a preferred embodiment, the cleavage siteis KLIP 8 (SEQ ID NO:143) which is cleavable by aspartic proteasesincluding chymosin. In a further preferred embodiment, an extramethionine residue is added to the N-terminus of the Apo AI polypeptideor pro-Apo AI polypeptide.

The invention further provides methods for the separation ofheterologous proteins from host cell components by partitioning of theoil body fraction and subsequent release of the heterologous protein viaspecific cleavage of the heterologous protein—oil body protein fusion.Optionally a cleavage site may be located upstream of the N-terminus anddownstream of the C-terminus of the heterologous polypeptide allowingthe fusion polypeptide to be cleaved and separated by phase separationinto its component peptides.

The nucleic acid sequence encoding apolipoprotein may be altered, toimprove expression levels for example, by optimizing the nucleic acidsequence in accordance with the preferred codon usage for a particularplant cell type which is selected for the expression of theapolipoprotein polypeptide, or by altering motifs known to destabilizemRNAs (see for example: PCT Patent Application 97/02352). Comparison ofthe codon usage of the nucleic acid sequence encoding the apolipoproteinpolypeptide with the codon usage of the plant cell type will enable theidentification of codons that may be changed. Construction of syntheticgenes by altering the codon usage is described in for example PCT PatentApplication 93/07278.

In a preferred embodiment, the nucleic acid sequence encodingapolipoprotein that is used is represented by SEQ. ID. NO 1, SEQ. ID.NO. 3 or SEQ. ID. NO. 5.

Certain genetic elements capable of enhancing expression of theapolipoprotein polypeptide may be used herein. These elements includethe untranslated leader sequences from certain viruses, such as the AMVleader sequence (Jobling and Gehrke, 1987, Nature, 325: 622-625) and theintron associated with the maize ubiquitin promoter (U.S. Pat. No.5,504,200). Generally the chimeric nucleic acid sequence will beprepared so that genetic elements capable of enhancing expression willbe located 5′ to the nucleic acid sequence encoding the apolipoproteinpolypeptide.

In accordance with the present invention the chimeric nucleic acidsequences comprising a promoter capable of controlling expression inplant linked to a nucleic acid sequence encoding an apolipoproteinpolypeptide can be integrated into a recombinant expression vector whichensures good expression in the cell. Accordingly, the present inventionincludes a recombinant expression vector comprising in the 5′ to 3′direction of transcription as operably linked components:

-   -   (i) a nucleic acid sequence capable of controlling expression in        plant cells; and    -   (ii) a nucleic acid sequence encoding an apolipoprotein        polypeptide;        wherein the expression vector is suitable for expression in a        plant cell. The term “suitable for expression in a plant cell”        means that the recombinant expression vector comprises the        chimeric nucleic acid sequence of the present invention linked        to genetic elements required to achieve expression in a plant        cell. Genetic elements that may be included in the expression        vector in this regard include a transcriptional termination        region, one or more nucleic acid sequences encoding marker        genes, one or more origins of replication and the like. In        preferred embodiments, the expression vector further comprises        genetic elements required for the integration of the vector or a        portion thereof in the plant cell's nuclear genome, for example        the T-DNA left and right border sequences which facilitate the        integration into the plant's nuclear genome in embodiments of        the invention in which plant cells are transformed using        Agrobacterium. In a further preferred embodiment said plant cell        is a plant seed cell.

As hereinbefore mentioned, the recombinant expression vector generallycomprises a transcriptional terminator which besides serving as a signalfor transcription termination further may serve as a protective elementcapable of extending the mRNA half life (Guarneros et al., 1982, Proc.Natl. Acad. Sci. USA, 79: 238-242). The transcriptional terminator isgenerally from about 200 nucleotides to about 1000 nucleotides and theexpression vector is prepared so that the transcriptional terminator islocated 3′ of the nucleic acid sequence encoding apolipoprotein.Termination sequences that may be used herein include, for example, thenopaline termination region (Bevan et al., 1983, Nucl. Acids. Res., 11:369-385), the phaseolin terminator (van der Geest et al., 1994, Plant J.6: 413-423), the arcelin terminator (Jaeger G D, et al., 2002, Nat.Biotechnol. 20:1265-8), the terminator for the octopine synthase genesof Agrobacterium tumefaciens or other similarly functioning elements.Transcriptional terminators may be obtained as described by An (An,1987, Methods in Enzym. 153: 292).

Pursuant to the present invention the expression vector may furthercontain a marker gene. Marker genes that may be used in accordance withthe present invention include all genes that allow the distinction oftransformed cells from non-transformed cells, including all selectableand screenable marker genes. A marker gene may be a resistance markersuch as an antibiotic resistance marker against, for example, kanamycin(U.S. Pat. No. 6,174,724), ampicillin, G418, bleomycin, hygromycin whichallows selection of a trait by chemical means or a tolerance markeragainst a chemical agent, such as the normally phytotoxic sugar mannose(Negrotto et al., 2000, Plant Cell Rep. 19: 798-803). Other convenientmarkers that may be used herein include markers capable of conveyingresistance against herbicides such as glyphosate (U.S. Pat. Nos.4,940,935; 5,188,642), phosphinothricin (U.S. Pat. No. 5,879,903) orsulphonyl ureas (U.S. Pat. No. 5,633,437). Resistance markers, whenlinked in close proximity to nucleic acid sequence encoding theapolipoprotein polypeptide, may be used to maintain selection pressureon a population of plant cells or plants that have not lost the nucleicacid sequence encoding the apolipoprotein polypeptide. Screenablemarkers that may be employed to identify transformants through visualinspection include β-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995,Plant Cell Rep., 14: 403).

Recombinant vectors suitable for the introduction of nucleic acidsequences into plants include Agrobacterium and Rhizobium based vectors,such as the Ti and Ri plasmids, including for example pBIN19 (Bevan,Nucl. Acid. Res., 1984, 22: 8711-8721), pGKB5 (Bouchez et al., 1993, C RAcad. Sci. Paris, Life Sciences, 316:1188-1193), the pCGN series ofbinary vectors (McBride and Summerfelt, 1990, Plant Mol. Biol.,14:269-276) and other binary vectors (e.g. U.S. Pat. No. 4,940,838).

The recombinant expression vectors of the present invention may beprepared in accordance with methodologies well known to those skilled inthe art of molecular biology. Such preparation will typically involvethe bacterial species Escherichia coli as an intermediairy cloning host.The preparation of the E. coli vectors as well as the planttransformation vectors may be accomplished using commonly knowntechnique's such as restriction digestion, ligation, gelectrophoresis,DNA sequencing, the Polymerase Chain Reaction (PCR) and othermethodologies. A wide variety of cloning vectors is available to performthe necessary steps required to prepare a recombinant expression vector.Among the vectors with a replication system functional in E. coli, arevectors such as pBR322, the pUC series of vectors, the M13mp series ofvectors, pBluescript etc. Typically, these cloning vectors contain amarker allowing selection of transformed cells. Nucleic acid sequencesmay be introduced in these vectors, and the vectors may be introduced inE. coli grown in an appropriate medium. Recombinant expression vectorsmay readily be recovered from cells upon harvesting and lysing of thecells. Further, general guidance with respect to the preparation ofrecombinant vectors may be found in, for example: Sambrook et al.,Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, Vol. 3.

Preparation of Plants Comprising Seed Capable of ExpressingApolipoprotein

In accordance with the present invention the chimeric nucleic acidsequence is introduced into a plant cell and the cells are grown intomature plants, wherein the plant expresses the apolipoproteinpolypeptide.

In accordance herewith any plant species or plant cell may be selected.Particular cells used herein include cells obtainable from Arabidopsisthaliana, Brazil nut (Betholettia excelsa); castor bean (Riccinuscommunis); coconut (Cocus nucifera); coriander (Coriandrum sativum);cotton (Gossypium spp.); groundnut (Arachis Hypogaea); jojoba(Simmondsia chinensis); linseed/flax (Linum usitatissimum); maize (Zeamays); mustard (Brassica spp. and Sinapis alba); oil palm (Elaeisguineeis); olive (Olea eurpaea); rapeseed (Brassica spp.); rice (Oryzasativa); safflower (Carthamus tinctorius); soybean (Glycine max); squash(Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticumaestivum); duckweed (Lemnaceae sp.) and sunflower (Helianthus annuus).

In accordance herewith in a preferred embodiment plant species or plantcells from oil seed plants are used. Oil seed plants that may be usedherein include peanut (Arachis hypogaea); mustard (Brassica spp. andSinapis alba); rapeseed (Brassica spp.); chickpea (Cicer arietinum);soybean (Glycine max); cotton (Gossypium hirsutum); sunflower(Helianthus annuus); (Lentil Lens culinaris); linseed/flax (Linumusitatissimum); white clover (Trifolium repens); olive (Olea eurpaea);oil palm (Elaeis guineeis); safflower (Carthamus tinctorius) and narbonbean (Vicia narbonesis).

In accordance herewith in a particularly preferred embodimentArabidopsis, flax or safflower is used.

Methodologies to introduce plant recombinant expression vectors into aplant cell, also referred to herein as “transformation”, are well knownto the art and typically vary depending on the plant cell that isselected. General techniques to introduce recombinant expression vectorsin cells include, electroporation; chemically mediated techniques, forexample CaCl₂ mediated nucleic acid uptake; particle bombardment(biolistics); the use of naturally infective nucleic acid sequences, forexample virally derived nucleic acid sequences, or Agrobacterium orRhizobium derived sequences, polyethylene glycol (PEG) mediated nucleicacid uptake, microinjection and the use of silicone carbide whiskers.

In preferred embodiments, a transformation methodology is selected whichwill allow the integration of the chimeric nucleic acid sequence in theplant cell's genome, and preferably the plant cell's nuclear genome. Inaccordance herewith this is considered particularly desirable as the useof such a methodology will result in the transfer of the chimericnucleic acid sequence to progeny plants upon sexual reproduction.Transformation methods that may be used in this regard includebiolistics and Agrobacterium mediated methods.

Transformation methodologies for dicotyledenous plant species are wellknown. Generally, Agrobacterium mediated transformation is used becauseof its high efficiency, as well as the general susceptibility by many,if not all, dicotyledenous plant species. Agrobacterium transformationgenerally involves the transfer of a binary vector, such as one of thehereinbefore mentioned binary vectors, comprising the chimeric nucleicacid sequence of the present invention from E. coli to a suitableAgrobacterium strain (e.g. EHA101 and LBA4404) by, for example,tri-parental mating with an E. coli strain carrying the recombinantbinary vector and an E. coli strain carrying a helper plasmid capable ofmobilizing the binary vector to the target Agrobacterium strain, or byDNA transformation of the Agrobacterium strain (Hofgen et al., Nucl.Acids. Res., 1988, 16:9877). Other techniques that may be used totransform dicotyledenous plant cells include biolistics (Sanford, 1988,Trends in Biotechn. 6:299-302); electroporation (Fromm et al., 1985,Proc. Natl. Acad. Sci. USA., 82:5824-5828); PEG mediated DNA uptake(Potrykus et al., 1985, Mol. Gen. Genetics, 199:169-177); microinjection(Reich et al., Bio/Techn., 1986, 4:1001-1004); and silicone carbidewhiskers (Kaeppler et al., 1990, Plant Cell Rep., 9:415-418) or inplanta transformation using, for example, a flower dipping methodology(Clough and Bent, 1998, Plant J., 16:735-743).

Monocotyledonous plant species may be transformed using a variety ofmethodologies including particle bombardment (Christou et al., 1991,Biotechn. 9:957-962; Weeks et al., Plant Physiol., 1993, 102:1077-1084;Gordon-Kamm et al., Plant Cell, 1990, 2:5603-618); PEG mediated DNAuptake (European Patents 0292 435; 0392 225) or Agrobacterium mediatedtransformation (Goto-Fumiyuki et al., 1999, Nature-Biotech. 17:282-286).

The exact plant transformation methodology may vary somewhat dependingon the plant species and the plant cell type (e.g. seedling derived celltypes such as hypocotyls and cotyledons or embryonic tissue) that isselected as the cell target for transformation. As hereinbeforementioned in a particularly preferred embodiment safflower is used. Amethodology to obtain safflower transformants is available in Baker andDyer (Plant Cell Rep., 1996, 16:106-110). Additional plant speciesspecific transformation protocols may be found in: Biotechnology inAgriculture and Forestry 46: Transgenic Crops I (Y. P. S. Bajaj ed.),Springer-Verlag, New York (1999), and Biotechnology in Agriculture andForestry 47: Transgenic Crops II (Y. P. S. Bajaj ed.), Springer-Verlag,New York (2001).

Following transformation, the plant cells are grown and upon theemergence of differentiating tissue, such as shoots and roots, matureplants are regenerated. Typically a plurality of plants is regenerated.Methodologies to regenerate plants are generally plant species and celltype dependent and will be known to those skilled in the art. Furtherguidance with respect to plant tissue culture may be found in, forexample: Plant Cell and Tissue Culture, 1994, Vasil and Thorpe Eds.,Kluwer Academic Publishers; and in: Plant Cell Culture Protocols(Methods in Molecular Biology 111), 1999, Hall Eds, Humana Press.

In one aspect, the present invention provides a method of obtainingplant seed comprising apolipoprotein. Accordingly, the present inventionprovides a method for obtaining plant seed comprising apolipoproteincomprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant seed cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell;    -   (c) growing the plant cell into a mature plant; and    -   (d) obtaining seed from said plant wherein the seed comprises        apolipoprotein.

The seeds may be used to obtain a population of progeny plants eachcomprising a plurality of seeds expressing apolipoprotein. In apreferred embodiment at least approximately 0.25% of the total seedprotein is apolipoprotein. More preferably least approximately 0.5% ofthe total seed protein is apolipoprotein. Most preferably at leastapproximately 1.0% of the total seed protein is apolipoprotein.

In preferred embodiments, a plurality of transformed plants is obtained,grown, and screened for the presence of the desired chimeric nucleicacid sequence, the presence of which in putative transformants may betested by, for example, growth on a selective medium, where herbicideresistance markers are used, by direct application of the herbicide tothe plant, or by Southern blotting. If the presence of the chimericnucleic acid sequence is detected, transformed plants may be selected togenerate progeny and ultimately mature plants comprising a plurality ofseeds comprising the desired chimeric nucleic acid sequence. Such seedsmay be used to isolate apolipoprotein or they may be planted to generatetwo or more subsequent generations. It will generally be desirable toplant a plurality of transgenic seeds to obtain a population oftransgenic plants, each comprising seeds comprising a chimeric nucleicacid sequence encoding apolipoprotein. Furthermore, it will generally bedesirable to ensure homozygosity in the plants to ensure continuedinheritance of the recombinant polypeptide. Methods for selectinghomozygous plants are well known to those skilled in the art. Methodsfor obtaining homozygous plants that may be used include the preparationand transformation of haploid cells or tissues followed by theregeneration of haploid plantlets and subsequent conversion to diploidplants for example by the treatment with colchine or other microtubuledisrupting agents. Plants may be grown in accordance with otherwiseconventional agricultural practices.

In another aspect, the present invention also provides plants capable ofsetting seed expressing apolipoprotein. In a preferred embodiment of theinvention, the plants capable of setting seed comprise a chimericnucleic acid sequence comprising in the 5′ to 3′ direction oftranscription:

-   -   (a) a first nucleic acid sequence capable of controlling        expression in a plant seed cell operatively linked to;    -   (b) a second nucleic acid sequence encoding an apolipoprotein        polypeptide, wherein the seed contains apolipoprotein.

In a preferred embodiment the chimeric nucleic acid sequence is stablyintegrated in the plant's nuclear genome.

In yet another aspect, the present invention provides plant seedsexpressing apolipoprotein. In a preferred embodiment of the presentinvention, the plant seeds comprise a chimeric nucleic acid sequencecomprising in the 5′ to 3′ direction of transcription:

-   -   (a) a first nucleic acid sequence capable of controlling        expression in a plant seed cell operatively linked to;    -   (b) a second nucleic acid sequence encoding an apolipoprotein        polypeptide.

The apolipoprotein polypeptide may be present in a variety of differenttypes of seed cells including, for example, the hypocotyls and theembryonic axis, including in the embryonic roots and embryonic leafs,and where monocotyledonous plant species, including cereals and corn,are used in the endosperm tissue.

Once the plants have been obtained the apolipoprotein protein may beextracted and obtained from the plant in a more or less pure form.

Accordingly, the present invention provides a method for preparingsubstantially pure apolipoprotein comprising:

-   -   (a) providing a chimeric nucleic acid construct comprising in        the 5′ to 3′ direction of transcription as operably linked        components:        -   (i) a nucleic acid sequence capable of controlling            expression in plant seed cells; and        -   (ii) a nucleic acid sequence encoding an apolipoprotein            polypeptide;    -   (b) introducing the chimeric nucleic acid construct into a plant        cell;    -   (c) growing the plant cell into a mature plant; and obtaining        seed from said plant wherein the seed comprises apolipoprotein;        and    -   (e) separating apolipoprotein from the plant seed constituents        to obtain substantially pure apolipoprotein.

In order to separate the apolipoprotein from the seed constituents,plant seeds may be ground using any comminuting process resulting in asubstantial disruption of the seed cell membrane and cell walls. Bothdry and wet milling conditions (U.S. Pat. No. 3,971,856; Lawhon et al.,1977, J. Am. Oil Chem. Soc., 63:533-534) may be used. Suitable millingequipment in this regard include colloid mills, disc mills, IKA mills,industrial scale homogenizers and the like. The selection of the millingequipment will depend on the seed type and throughput requirements.Solid seed contaminant such as seed hulls, fibrous materials,undissolved carbohydrates, proteins and other water insolublecontaminants may be removed from the seed fraction using for examplesize-exclusion based methodologies, such as filtering or gravitationalbased processes such as centrifugation. In preferred embodiments, theuse of organic solvents commonly used in oil extraction, such as hexane,is avoided because such solvents may damage the apolipoproteinpolypeptide. As hereinbefore mentioned in preferred embodiments of thepresent invention the apoliprotein is prepared in a manner that permitsassociation of the apolipoprotein polypeptide with seed oil bodies.Accordingly, seed oil bodies comprising the apolipoprotein may beprepared following comminuting of the seed using for example themethodologies detailed in U.S. Pat. No. 6,146,645. Thus the presentinvention also includes substantially pure oil bodies comprisingapolipoprotein obtained from plant seed. The oil bodies may be used as arefined plant seed fraction to further purify apolipoprotein.Substantially pure apolipoprotein may be recovered from seed using avariety of additional purification methodologies such as centrifugationbased techniques; size exclusion based methodologies, including forexample membrane ultrafiltration and crossflow ultrafiltration; andchromatographic techniques, including for example ion-exchangechromatography, size exclusion chromatography, affinity chromatography,high performance liquid chromatography (HPLC), fast protein liquidchromatography (FPLC), hydrophobic interaction chromatography and thelike. Generally, a combination of such techniques will be used to obtainsubstantially pure apolipoprotein. A preferred methodology to obtainsubstantially pure apoliprotein in its native form from transgenic plantseeds is further detailed in Example 6 herein. Thus the presentinvention also includes substantially pure apolipoprotein obtained froma plant.

Pharmaceutical apolipoprotein formulations may be prepared from thepurified apolipoprotein and such formulations may be used to treatvascular diseases. Generally the purified apolipoprotein will be admixedwith a pharmaceutically acceptable carrier or diluent in amountssufficient to exert a therapeutically useful effect in the absence ofundesirable side effects on the patient treated. To formulate anapolipoprotein composition, the weight fraction of apolipoprotein isdissolved, suspended, dispersed or otherwise mixed in a selected carrieror diluent at an effective concentration such that the treated conditionis ameliorated. The pharmaceutical apolipoprotein formulations arepreferably formulated for single dosage administration. Therapeuticallyeffective doses for the parenteral delivery of human apolipoprotein arewell known to the art. Where apolipoprotein analogs are used or othermodes of delivery are used therapeutically effective doses may bereadily empirically determined by those of skill in the art using knowntesting protocols or by extrapolation of in-vivo or in-vitro test data.It is understood however that concentrations and dosages may vary inaccordance with the severity of the condition alleviated. It is furtherunderstood that for any particular subject, specific dosage regimens maybe adjusted over time according to individual judgement of the personadministering or supervising administration of the formulations.

Pharmaceutical solutions or suspensions may include for example asterile diluent such as, for example, water, lactose, sucrose, dicalciumphosphate, or carboxymethyl cellulose. Carriers that may be used includewater, saline solution, aqueous dextrose, glycerol, glycols, ethanol andthe like, to thereby form a solution or suspension. If desired thepharmaceutical compositions may also contain non-toxic auxiliarysubstances such a wetting agents; emulsifying agents; solubilizingagents; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid (EDTA); pHbuffering agents such as actetate, citrate or phosphate buffers; andcombinations thereof.

The final formulation of the apolipoprotein preparation will generallydepend on the mode of apolipoprotein delivery. The apolipoproteinprepared in accordance with the present invention may be delivered inany desired manner; however parenteral, oral and nasal forms of deliveryare considered the most likely used modes of delivery. Parenteralpreparations can be enclosed in ampoules, disposable syringes or singleor multiple dose vials made of glass, plastic or other such suitablematerials.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Construction of Apolipoprotein A-I Clones

Apo10

Apo10 (SEQ ID NO:144) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(C). As seen inFIG. 2, the Apo10 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:145) between mature Apo AI and GFP. To construct this cloneforward primer 1186 (SEQ ID NO:146 (5′-GGATCCCCtTGGCTAGTAAAGG-3′)removed a NcoI site from the start of GFP (template derived from thevector pVS-GFP). Reverse primer 1187 (SEQ ID. NO:147)(5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI, XbaI andHindIII sites after the stop codon. The PCR fragment was ligated intothe EcoRI cloning vector Topo (Invitrogen) creating plasmid G1 (FIG.3(A)). Forward primer 1190 (SEQ ID NO:148)(5′-CCATGGggCGGCATTTCTGGCAGCAAGATG-3′) amplifies the mature sequence ofApo AI and adds a NcoI site to the start of gene. Reverse primer 1189(SEQ ID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stopcodon of the gene and adds a BamHI site to assist in creating a in-frametranslation fusion with GFP (Plasmid G1). The template for these primerswas a pKS+ based vector (Strategene) containing the entire codingsequence for human Apo AI gene. The PCR fragment was ligated into theEcoRI site of the Topo cloning vector (Invitrogen) creating Plasmid M2.Plasmid M2 contained the mature sequence of Apo AI was cut withrestriction enzymes NcoI and BamHI. Plasmid G′1 contains the GFP codingsequence and was cut with BamHI and XbaI (see FIG. 3(B)). The fragmentsof M2 and G′1 were ligated together into the NcoI and XbaI sites of theplasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2M4. Plasmid 2M4was cut with NcoI and HindIII to remove the Apo AI-GFP fusion cassetteand the fragments were used subsequently to clone into the NcoI/HindIIIsites of binary vector pSBS4006 (see FIG. 3(C)). Note that this plasmidcontains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) and apat gene conferring host plant phosphinothricine resistance (Wohllebenet al., 1988, Gene 70:25-37) driven by the ubiquitin promoter/terminatorfrom Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium.

Apo11

Apo11 (SEQ ID NO:150) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(C). As seen inFIG. 2, the Apo11 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:151) between pro-Apo AI and GFP. To construct this clone, forwardprimer 1186 (SEQ ID NO:146) (5′-GGATCCCCtTGGCTAGTAAAGG-3′) removed aNcoI site from the start of GFP (template derived from the vectorpVS-GFP). Reverse primer 1187 (SEQ ID NO:147)(5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI, XbaI andHindIII sites after the stop codon. The PCR fragment was ligated intothe EcoRI cloning vector Topo (Invitrogen) creating plasmid G1 (FIG.3(A)). Forward primer 1191 (SEQ ID NO:152)(5′-CCATGGATGAACCCCCCCAGAGCCCCTG-3′) amplifies the pro-sequence of ApoAI and adds a NcoI site to the start of gene. Reverse primer 1189 (SEQID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stop codonof the gene and adds a BamHI site to assist in creating a translationfusion with GFP (Plasmid G1). The template for these primers was a pKS+based vector (Strategene) containing the entire coding sequence forhuman Apo AI gene. The PCR fragments were each separately ligated intothe EcoRI site of the Topo cloning vector (Invitrogen) creating PlasmidP2. Plasmid P2 contained the proApo AI sequence was cut with restrictionenzymes NcoI and BamHI. Plasmid G′1 contains the GFP coding sequence andwas cut with BamHI and XbaI (see FIG. 3(B)). The fragments of P2 and G′1were ligated together into the NcoI and XbaI sites of the plasmidSBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid 2P5 was cutwith NcoI and HindIII to remove the pro-Apo AI-GFP fusion cassette andthe fragments were used subsequently to clone into the NocI/HindIIIsites binary vector pSBS4006 (see FIG. 3(C). Note that this plasmidcontains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) and apat gene conferring host plant phosphinothricine resistance (Wohllebenet al., 1988, Gene 70:25-37) driven by the ubiquitin promoter/terminatorfrom Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium.

Apo12

Apo12 (SEQ ID NO:153) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(C). As seen inFIG. 2, the Apo12 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:154) between an oleosin (van Rooijen, G. J., et al. Plant Mol.Biol. 18 (6), 1177-1179 (1992)), mature Apo AI and GFP. To constructthis clone forward primer 1186 (SEQ ID NO:146)(5′-GGATCCCCtTGGCTAGTAAAGG-3′) removed a NcoI site from the start of GFP(template derived from the vector pVS-GFP). Reverse primer 1187 (SEQ IDNO:147) (5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI,XbaI and HindIII sites after the stop codon. The PCR fragment wasligated into the EcoRI cloning vector Topo (Invitrogen) creating plasmidG1 (FIG. 3(A)). Forward primer 1190 (SEQ ID NO: 148)(5′-CCATGGggCGGCATTTCTGGCAGCAAGATG-3′) amplifies the mature sequence ofApo AI and adds a NcoI site to the start of gene. Reverse primer 1189(SEQ ID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stopcodon of the gene and adds a BamHI site to assist in creating a in-frametranslation fusion with GFP (Plasmid G1). The template for these primerswas a pKS+ based vector (Strategene) containing the entire codingsequence for human Apo AI gene. The PCR fragment was ligated into theEcoRI site of the Topo cloning vector (Invitrogen) creating Plasmid M2.Plasmid M2 contained the mature sequence of Apo AI was cut withrestriction enzymes NcoI and BamHI. Plasmid G′1 contains the GFP codingsequence and was cut with BamHI and XbaI (see FIG. 3(B)). The fragmentsof M2 and G′1 were ligated together into the NcoI and XbaI sites of theplasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2M4. Plasmid 2M4was cut with NcoI and HindIII to remove the Apo AI-GFP fusion cassetteand the fragments were used subsequently to clone into the NcoI/HindIIIsites of binary vector pSBS4008 (see FIG. 3(C)). Note that this plasmidcontains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the Arabidopsis oleosin gene (van RooijenG. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) for fusion withthe Apo AI/GFP fusion. The plasmid also contains a pat gene conferringhost plant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37)) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium.

Apo13

Apo13 (SEQ ID NO:155) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(C). As seen inFIG. 2, the Apo13 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:156) between oleosin, pro-Apo AI and GFP. To construct this clone,forward primer 1186 (SEQ ID NO:146) (5′-GGATCCCCtTGGCTAGTAAAGG-3′)removed a NcoI site from the start of GFP (template derived from thevector pVS-GFP). Reverse primer 1187 (SEQ ID NO:147)(5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI, XbaI andHindIII sites after the stop codon. The PCR fragment was ligated intothe EcoRI cloning vector Topo (Invitrogen) creating plasmid G1 (FIG.3(A)). Forward primer 1191 (SEQ ID NO:152)(5′-CCATGGATGAACCCCCCCAGAGCCCCTG-3′) amplifies the pro-sequence of ApoAI and adds a NcoI site to the start of gene. Reverse primer 1189 (SEQID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stop codonof the gene and adds a BamHI site to assist in creating a translationfusion with GFP (Plasmid G1). The template for these primers was a pKS+based vector (Strategene) containing the entire coding sequence forhuman Apo AI gene. The PCR fragments were each separately ligated intothe EcoRI site of the Topo cloning vector (Invitrogen) creating PlasmidP2. Plasmid P2 contained the pro-Apo AI sequence was cut withrestriction enzymes NcoI and BamHI. Plasmid G′1 contains the GFP codingsequence and was cut with BamHI and XbaI (see FIG. 3(B)). The fragmentsof P2 and G′1 were ligated together into the NcoI and XbaI sites of theplasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid 2P5was cut with NcoI and HindIII to remove the pro-Apo AI-GFP fusioncassette and the fragments were used subsequently to clone into theNocI/HindIII sites of binary vector pSBS4008 (see FIG. 3(C)). Note thatthis plasmid contains the β-phaseolin promoter/terminator from Phaseolusvulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the Arabidopsis oleosin gene (van RooijenG. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) for fusion withthe Apo AI/GFP fusion. The plasmid also contains a pat gene conferringhost plant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium.

Apo15

Apo15 (SEQ ID NO:157) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(D). As seen inFIG. 2, the Apo15 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:158) between mature Apo AI and GFP. The fusion protein wastargeted for expression through the secretory pathway using the tobaccopathogen related sequence (PRS) signal peptide (Sijmons et al., 1990,Bio/technology, 8:217-221). To construct this clone forward primer 1186(SEQ ID NO:146) (5′-GGATCCCCtTGGCTAGTAAAGG-3′) removed a NcoI site fromthe start of GFP (template derived from the vector pVS-GFP). Reverseprimer 1187 (SEQ ID NO: 147)(5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI, XbaI andHindIII sites after the stop codon. The PCR fragment was ligated intothe EcoRI cloning vector Topo (Invitrogen) creating plasmid G1 (FIG.3(A)). Forward primer 1190 (SEQ ID NO:148)(5′-CCATGGggCGGCATTTCTGGCAGCAAGATG-3′) amplifies the mature sequence ofApo AI and adds a NcoI site to the start of gene. Reverse primer 1189(SEQ ID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stopcodon of the gene and adds a BamHI site to assist in creating a in-frametranslation fusion with GFP (Plasmid G1). The template for these primerswas a pKS+ based vector (Strategene) containing the entire codingsequence for human Apo AI gene. The PCR fragment was ligated into theEcoRI site of the Topo cloning vector (Invitrogen) creating Plasmid M2.Plasmid M2 contained the mature sequence of Apo AI was cut withrestriction enzymes NcoI and BamHI. Plasmid G′1 contains the GFP codingsequence and was cut with BamHI and XbaI (see FIG. 3(B)). The fragmentsof M2 and G′1 were ligated together into the NcoI and XbaI sites of theplasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2M4. Plasmid 2M4was cut with NcoI and HindIII to remove the Apo AI-GFP fusion cassetteand the fragments were used subsequently to clone into the NcoI/HindIIIsites of binary vector pSBS4011 (see FIG. 3(D)). Note that this plasmidcontains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) fused tothe PRS signal peptide. The plasmid also contains a pat gene conferringhost plant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684)) fortransformation into Agrobacterium.

Apo16

Apo16 (SEQ ID NO:159) is a clone designed to express in a seed-specificmanner which is constructed as per FIGS. 3(A), 3(B) and 3(D). As seen inFIG. 2, the Apo16 clone consists of a seed-specific promoter andterminator (phaseolin) driving the expression of a fusion protein (SEQID NO:160) between pro-Apo AI and GFP. The fusion protein was targetedfor expression through the secretory pathway using the tobacco pathogenrelated sequence (PRS) signal peptide (Sijmons et al., 1990,Bio/technology, 8:217-221). To construct this clone, forward primer 1186(SEQ ID NO:146) (5′-GGATCCCCtTGGCTAGTAAAGG-3′) removed a NcoI site fromthe start of GFP (template derived from the vector pVS-GFP). Reverseprimer 1187 (SEQ ID NO: 147)(5′-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3′) added PstI, XbaI andHindIII sites after the stop codon. The PCR fragment was ligated intothe EcoRI cloning vector Topo (Invitrogen) creating plasmid G1 (FIG.3(A)). Forward primer 1191 (SEQ ID NO:152)(5′-CCATGGATGAACCCCCCCAGAGCCCCTG-3′) amplifies the pro-sequence of ApoAI and adds a NcoI site to the start of gene. Reverse primer 1189 (SEQID NO:149) (5′-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stop codonof the gene and adds a BamHI site to assist in creating a translationfusion with GFP (Plasmid G1). The template for these primers was a pKS+based vector (Strategene) containing the entire coding sequence forhuman Apo AI gene. The PCR fragments were each separately ligated intothe EcoRI site of the Topo cloning vector (Invitrogen) creating PlasmidP2. Plasmid P2 contained the pro-Apo AI sequence was cut withrestriction enzymes NcoI and BamHI. Plasmid G′1 contains the GFP codingsequence and was cut with BamHI and XbaI (see FIG. 3(B)). The fragmentsof P2 and G′1 were ligated together into the NcoI and XbaI sites of theplasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid 2P5was cut with NcoI and HindIII to remove the pro-Apo AI-GFP fusioncassette and the fragments were used subsequently to clone into theNcoI/HindIII sites of binary vector pSBS4011 (see FIG. 3(D)). Note thatthis plasmid contains the β-phaseolin promoter/terminator from Phaseolusvulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)fused to the PRS signal peptide.

The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium.

Apo17

Apo17 (SEQ ID NO:161) is clone designed to express in a constitutivemanner which is constructed as per FIG. 4(A). As seen in FIG. 2, theApo17 clone consists of a constitutive promoter and terminator(ubiquitin) driving the expression of a fusion protein (SEQ ID NO:162)between Apo AI and GFP. To construct this clone, plasmid 2M4 (see FIG.3(B)) was cut with NcoI and PstI to remove the Apo AI-GFP fusioncassette and the fragment was ligated into the NcoI and PstI sites ofthe plasmid pKUO3′ to create plasmid KU2M4. The pKUO3′ plasmid containsa Brassica napus oleosin gene which is removed when the vector is cutwith NcoI and PstI resulting in the Apo AI/GFP fusion construct underthe control of a parsley ubiquitin promoter and ubiquitin terminator(Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684). The KU2M4plasmid was cut with KpnI and ligated into the KpnI site of the binaryvector SBS3000 (FIG. 4. Note that this plasmid contains a pat geneconferring host plant phosphinothricine resistance (Wohlleben et al.,1988, Gene 70:25-37) driven by the ubiquitin promoter/terminator fromPetroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium. The Apo17 clonecontains a mature Apo AI-GFP translational fusion and is targeted to thecytosol.

Apo18a

Apo18a (SEQ ID NO:163) is clone designed to express in a constitutivemanner which is constructed as per FIG. 4. As seen in FIG. 2, the Apo18aclone consists of a constitutive promoter and terminator (ubiquitin)driving the expression of a fusion protein (SEQ ID NO:164) betweenpro-Apo AI and GFP. To construct this clone, plasmid 2P5 (see FIG. 3(B))was cut with NcoI and PstI to remove the pro-Apo AI-GFP fusion cassetteand the fragment was ligated into the NcoI and PstI sites of the plasmidpKUO3′ to create plasmid KU2P5. The pKUO3′ plasmid contains a Brassicanapus oleosin gene which is removed when the vector is cut with NcoI andPstI resulting in the pro-Apo AI/GFP fusion construct under the controlof a parsley ubiquitin promoter and ubiqutin terminator (Kawalleck, P.et al., 1993, Plant Mol. Biol. 21:673-684). The KU2P5 plasmid was cutwith KpnI and ligated into the KpnI site of the binary vector SBS3000(FIG. 4. Note that this plasmid contains a pat gene conferring hostplant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37)) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. The Apo18a clone contains a pro-ApoAI-GFP translational fusion and is targeted to the cytosol.

Apo18b

Apo18b (SEQ ID NO:163) is clone designed to express in a constitutivemanner which is constructed as per FIG. 4(B). As seen in FIG. 2, theApo18b clone consists of a constitutive promoter and terminator(ubiquitin) driving the expression of a fusion protein (SEQ ID NO:164)between pro-Apo AI and GFP. Note that clones Apo18a and Apo18b bothconsist have a constitutive promoter (ubiquitin) driving the expressiona fusion protein (SEQ ID NO:164) between pro-Apo AI and GFP. Thedifference between the 2 clones is that Apo18a is inserted into the KpnIsite of binary vector pSBS3000 (which contains the pat gene conferringhost plant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. In contrast, Apo18b is inserted intothe KpnI site of binary vector pSBS5001 (which contains the pmi gene(Miles et al., 1984, Gene 21:41-48), encoding for phosphomannoseisomerase which allows for positive selection on mannose containingselection media. The pmi gene is under the control of the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium. Toconstruct this clone, the Apo18a binary vector was cut with KpnI removethe pro-Apo AI-GFP fusion cassette and the fragment was ligated into theKpnI site of the plasmid SBS5001 for expression in Agrobacterium. TheApo18b clone contains a pro-Apo AI-GFP translational fusion and istargeted to the cytosol.

Apo19

Apo19 (SEQ ID NO:165) is clone designed to express in a constitutivemanner which is constructed as per FIGS. 5(A) and 5(B). As seen in FIG.2, the Apo19 clone consists of a constitutive promoter and terminator(ubiquitin) driving the expression of a fusion protein (SEQ ID NO:166)between Apo AI and GFP. The fusion protein was targeted for expressionthrough the secretory pathway using the tobacco pathogen relatedsequence (PRS) signal peptide (Sijmons et al., 1990, Bio/technology,8:217-221). To construct this clone, Apo15 is used as a template.Forward primer 1177 (SEQ ID NO:167)(5′-GCAGCATTCATGAACTTCCTTAAGTCTTTCC-3′) amplifies the start of the plantpresequence (PRS) which contains a BspHI site at the start codon.Reverse primer 1178 (SEQ ID NO:168)(5′-GGTGGTGGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stop codon ofthe gene and adds a BamHI site to assist in creating an in-frametranslation fusion with GFP. Extra bases were left on the ends of bothprimers to facilitate restriction enzyme digestion. The G1 plasmid (seeFIG. 3(A)) was digested with BamHI and PstI for ligation into pubiP+iS3′which contains the ubiquitin promoter and terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684). ThePRS-Apo AI PCR fragment was digested with BspHI and BamHI and ligatedwith the G1 fragment into the plasmid pubiP+iS3′ to create plasmid 19-6.Plasmid 19-6 was digested with EcoRI to remove the expression cassetteand the cassette was then ligated into the plasmid pKUO3′K (FIG. 4(A))between the EcoRI sites (removing the existing cassette), creatingplasmid KU19-6. KU19-6 was digested with KpnI and the fragment wasligated into the KpnI sites of the plasmid SBS3000 (FIG. 5(B)). Notethat this plasmid contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. The Apo19 clone contains a mature ApoAI-GFP translational fusion, respectively, targeted to the secretorypathway.

Apo20

Apo20 (SEQ ID NO:169) is clone designed to express in a constitutivemanner which is constructed as per FIGS. 5(A) and 5(B). As seen in FIG.2, the Apo20 clone consists of a constitutive promoter and terminator(ubiquitin) driving the expression of a fusion protein (SEQ ID NO:170)between pro-Apo AI and GFP. The fusion protein was targeted forexpression through the secretory system using the tobacco pathogenrelated sequence (PRS) signal peptide (Sijmons et al., 1990,Bio/technology, 8:217-221). To construct this clone, Apo16 is used as atemplate. Forward primer 1177 (SEQ ID NO:167)(5′-GCAGCATTCATGAACTTCCTTAAGTCTTTCC-3′) amplifies the start of the plantpresequence (PRS) which contains a BspHI site at the start codon.Reverse primer 1178 (SEQ ID NO:168)(5′-GGTGGTGGATCCcCTGGGTGTTGAGCTTCTTAGTG-3′) removes the stop codon ofthe gene and adds a BamHI site to assist in creating an in-frametranslation fusion with GFP. Extra bases were left on the ends of bothprimers to facilitate restriction enzyme digestion. The G1 plasmid (seeFIG. 3(A)) was digested with BamHI and PstI for ligation into pubiP+iS3′which contains the ubiquitin promoter and terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684). ThePRS-pro-Apo AI PCR fragment was digested with BspHI and BamHI andligated with the G1 fragment into the plasmid pubiP+iS3′ to createplasmid 20-11. Plasmid 20-11 was digested with EcoRI to remove theexpression cassette and the cassette was then ligated into the plasmidpKUO3′K (FIG. 4(A)) between the EcoRI sites (removing the existingcassette), creating plasmid KU20-11. KU20-11 was digested with KpnI andthe fragment was ligated into the KpnI sites of the plasmid SBS3000(FIG. 5(B). Note that this plasmid contains a pat gene conferring hostplant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37)) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. The Apo20 clone contains a pro-ApoAI-GFP translational fusion, respectively, targeted to the secretorypathway.

Apo21

Apo21 (SEQ ID NO:171) is a seed-preferred clone which is constructed asper FIG. 6. As seen in FIG. 2, the Apo21 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of Apo AI (SEQ ID NO:172). To construct this clone forwardprimer 1203 (SEQ ID NO:173) (5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′)adds a NcoI site to the start of mature Apo AI. Reverse primer 1206 (SEQID NO: 174) (5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGAGCG-3′) adds a HindIII site after the stop codon and adds a silentmutation to remove the second XhoI site. Both primers contained extrabases on the 5′ ends to facilitate restriction enzyme digestion. The PCRfragment was digested with NcoI and HindIII and ligated into the plasmidSBS2090 creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was digested withNcoI and HindIII and the Apo AI fragment was ligated into theNcoI/HindIII sites of SBS4006 (FIG. 3(C)). SBS4006 contains theβ-phaseolin promoter/terminator from Phaseolus vulgaris (Slightom etal., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) and a pat geneconferring host plant phosphinothricine resistance (Wohlleben et al.,1988, Gene 70:25-37)) driven by the ubiquitin promoter/terminator fromPetroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium. Apo21 is a clone forseed-specific targeting of Apo AI to the cytosol.

Apo22

Apo22 (SEQ ID NO:175) is a seed-preferred clone which is constructed asper FIG. 7. As seen in FIG. 2, the Apo22 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of pro-Apo AI (SEQ ID NO:176). To construct this cloneforward primer 1201 (SEQ ID NO: 177)(5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′) adds an NcoI site to thestart of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090(FIG. 3(B)) creating plasmid 4-2 (FIG. 7). Plasmid 4-2 was digested withNcoI and HindIII and the pro-Apo AI fragment was ligated into theNcoI/HindIII sites of SBS4006 (FIG. 3(C)). SBS4006 contains theβ-phaseolin promoter/terminator from Phaseolus vulgaris (Slightom etal., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) and a pat geneconferring host plant phosphinothricine resistance (Wohlleben et al.,1988, Gene 70:25-37) driven by the ubiquitin promoter/terminator fromPetroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684)) for transformation into Agrobacterium. Apo22 is a clone forseed-specific targeting of pro-Apo AI to the cytosol.

Apo23

Apo23 (SEQ ID NO:178) is a seed-preferred clone which is constructed asper FIG. 6. As seen in FIG. 2, the Apo23 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/Apo AI (SEQ ID NO:179). To construct this cloneforward primer 1203 (SEQ ID NO: 173)(5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′) adds an NcoI site to thestart of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was digested with NcoI andHindIII and the Apo AI fragment was ligated into the NcoI/HindIII sitesof SBS4008 (FIG. 3(C)). SBS4008 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol.Biol. 18 (6), 1177-1179) and a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37))driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684)) fortransformation into Agrobacterium. Apo23 is a clone for seed-specifictargeting of oleosin/Apo AI to the oil bodies.

Apo24

Apo24 (SEQ ID NO:180) is a seed-preferred clone which is constructed asper FIG. 7. As seen in FIG. 2, the Apo24 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/pro-Apo AI (SEQ ID NO:181). To construct thisclone forward primer 1201 (SEQ ID NO: 177)(5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′) adds an NcoI site to thestart of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090creating plasmid 4-2 (FIG. 7). Plasmid 4-2 was digested with NcoI andHindIII and the pro-Apo AI fragment was ligated into the NcoI/HindIIIsites of SBS4008 (FIG. 3(C)). SBS4008 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol.Biol. 18 (6), 1177-1179) and a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo24 is a clone for seed-specifictargeting of oleosin/pro-Apo AI to the oil bodies.

Apo25

Apo25 (SEQ ID NO:182) is a seed-preferred clone which is constructed asper FIG. 6. As seen in FIG. 2, the Apo25 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/klip8/met/Apo AI (SEQ ID NO:183). This constructhas a klip8 cleavage sequence (SEQ ID NO:143) to facilitate cleavage ofthe fusion protein with chymosin. To construct this clone forward primer1203 (SEQ ID NO:173) (5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′) adds anNcoI site to the start of mature Apo AI. Reverse primer 1206 (SEQ IDNO:174) (5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′)adds a HindIII site after the stop codon and adds a silent mutation toremove the second XhoI site. Both primers contained extra bases on the5′ ends to facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090(FIG. 3(B)) creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was digested withNcoI and HindIII and the Apo AI fragment was ligated into theNcoI/HindIII sites of SBS4010 (FIG. 7). SBS4010 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol.Biol. 18 (6), 1177-1179) and a klip8 cleavage site. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37)) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium.

Apo25 is a clone for seed-specific targeting of oleosin/klip8/met/Apo AIto the oil bodies and purification using the klip8 cleavage sequence.

Apo26

Apo26 (SEQ ID NO:184) is a seed-preferred clone which is constructed asper FIG. 7. As seen in FIG. 2, the Apo26 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/klip8/met/pro-Apo AI (SEQ ID NO:185). Thisconstruct has a klip8 cleavage sequence (SEQ ID NO:143) to facilitatecleavage of the fusion protein with chymosin. To construct this cloneforward primer 1201 (SEQ ID NO:177)(5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′) adds an NcoI site to thestart of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090(FIG. 3(B)) creating plasmid 4-2 (FIG. 7). Plasmid 4-2 was digested withNcoI and HindIII and the pro-met-Apo AI fragment was ligated into theBspHI/HindIII sites of SBS4010 (FIG. 7). SBS4010 contains theβ-phaseolin promoter/terminator from Phaseolus vulgaris (Slightom etal., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling theexpression of the Arabidopsis oleosin gene (van Rooijen G. J. et al.1992, Plant Mol. Biol. 18 (6), 1177-1179) and a klip8 cleavage site. Theplasmid also contains a pat gene conferring host plant phosphinothricineresistance (Wohlleben et al., 1988, Gene 70:25-37) driven by theubiquitin promoter/terminator from Petroselinum crispum (Kawalleck etal., 1993, Plant. Mol. Bio., 21:673-684) for transformation intoAgrobacterium. Apo26 is a clone for seed-specific targeting ofoleosin/klip8/met/pro-Apo AI to the oil bodies and purification usingthe klip8 cleavage sequence.

Apo27

Apo27 (SEQ ID NO:186) is a seed-preferred clone which is constructed asper FIG. 8(A). As seen in FIG. 2, the Apo27 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/klip8/Apo AI (SEQ ID NO:187). Apo27 was targetedfor expression to the oil bodies using the Arabidopsis oleosin sequence(van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) andhas a klip8 cleavage sequence (SEQ ID NO:143) to facilitate cleavage ofthe fusion protein with chymosin. To construct this clone forward primer1200 (SEQ ID NO:188) (5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′)adds an XhoI site and extra nucleotides to facilitate in-frame cloninginto the klip8 (SEQ ID NO:143) cleavage sequence to the start of pro-ApoAI. Reverse primer 1206 (SEQ ID NO: 174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. The template for these primers was the Apo33plasmid which contains the pro-form of Apo AI without internal XhoIsites and additional Met residue. The PCR fragments were cut with XhoIand ligated into the XhoI/EcoRV sites of pKS+ creating the plasmids 6-3.Plasmid 6-3 was cut with XhoI and HindIII and ligated into theXhoI/HindIII sites of binary bector SBS4010 (FIG. 7.) Note SBS4010contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the Arabidopsis oleosin gene (van RooijenG. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) and a klip8cleavage site. The plasmid also contains a pat gene conferring hostplant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37)) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684)) fortransformation into Agrobacterium. Apo27 is a clone for seed-specifictargeting of Apo AI to oil bodies and purification with cleavagesequence klip8.

Apo27M

Apo27M (SEQ ID NO:189) is a seed-preferred clone which is constructed asper FIG. 8(B) As seen in FIG. 2, the Apo27M clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/klip8/met/Apo AI-M (SEQ ID NO:190). This constructhas a klip8 cleavage sequence (SEQ ID NO:143) to facilitate cleavage ofthe fusion protein with chymosin. To construct this clone, forwardprimer 1202 (5′-GCAGCACTCGAGcaagttcGATGAACCCCCCCAGAGCCC-3′) (SEQ IDNO:191) adds an XhoI site and extra nucleotides to facilitate in-framecloning into the klip8 cleavage sequence to the start of mat-Apo AI.Forward primer 1225 (5′-CGCCAGtGCTTGGCCGCGCGCCTTG-3′) (SEQ ID NO:192) isa blunt ended primer which makes a base pair mutation from C to T tochange an Arg residue into a Cys residue. Reverse primer 1206(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) (SEQ IDNO:174) adds a HindIII site after the stop codon and adds a silentmutation to remove the second XhoI site. Both primers contained extrabases on the 5′ ends to facilitate restriction enzyme digestion. Thetemplate for these primers was the plasmid P-10 which already had itsXhoI sites mutated. The double-stranded template was removed by DpnIdigestion. The PCR fragment was digested with XhoI and HindIII andligated into the plasmid pKS+ XhoI/HindIII sites creating plasmid ApoM.ApoM was digested with XhoI and HindIII and the fragment was ligatedinto the XhoI/HindIII sites of the plasmid SBS4010 (FIG. 7). SBS4010contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the Arabidopsis oleosin gene (van RooijenG. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) and a klip8cleavage site. The plasmid also contains a pat gene conferring hostplant phosphinothricine resistance (Wohlleben et al., 1988, Gene70:25-37) driven by the ubiquitin promoter/terminator from Petroselinumcrispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo27M is a clone for seed-specifictargeting of oleosin/klip8/met/Apo AI-M to the oil bodies andpurification using the klip8 cleavage sequence.

Apo 28

Apo28 (SEQ ID NO:193) is a seed-preferred clone which is constructed asper FIG. 9. As seen in FIG. 2, the Apo28 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of oleosin/klip8/pro-Apo AI (SEQ ID NO:194). To constructthis clone forward primer 1200 (SEQ ID NO: 188)(5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′) adds an XhoI site andextra nucleotides to facilitate in-frame cloning into the klip8 cleavagesequence to the start of pro-Apo AI. Forward primer 1205 (SEQ ID NO:195)(5′-CCAAGCCCGCGCTaGAGGACCTCCG-3′) is a blunt ended primer which adds asilent mutation to remove the first XhoI site. Reverse primer 1206 (SEQID NO: 174) (5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGAGCG-3′) adds a HindIII site after the stop codon and adds a silentmutation to remove the second XhoI site. Both primers contained extrabases on the 5′ ends to facilitate restriction enzyme digestion. Thetemplate for these primers was a pKS+ based vector (Stratagene) whichcontained the entire coding sequence for human Apo AI gene. Thedouble-stranded template was removed by DpnI digestion. The PCR fragmentwas digested with XhoI and HindIII and ligated into the plasmid pKS+XhoI/HindIII sites creating plasmid P-10. P-10 was digested with XhoIand HindIII and the fragment was ligated into the XhoI/HindIII sites ofthe plasmid SBS4010 (FIG. 7). Note SBS4010 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol.Biol. 18 (6), 1177-1179) and a klip8 cleavage site. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37)) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684)) for transformation into Agrobacterium.Apo28 is a pro-Apo AI clone targeted to oil bodies and able to becleaved at the klip8 sequence.

Apo29

Apo29 (SEQ ID NO:196) is a seed-preferred clone which is constructed asper FIG. 6. As seen in FIG. 2, the Apo29 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS-Apo AI (SEQ ID NO:197). Apo29 was targeted forexpression to the through the secretory pathway using the tobaccopathogen related sequence (PRS) signal peptide (Sijmons et al., 1990,Bio/technology, 8:217-221). To construct this clone forward primer 1203(SEQ ID NO:173) (5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′) adds an NcoIsite to the start of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The PCR fragment wasdigested with NcoI and HindIII and ligated into the plasmid SBS2090creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was digested with NcoI andHindIII and the Apo AI fragment was ligated into the NcoI/HindIII sitesof SBS4011 (FIG. 3D). SBS4011 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol.Biol. 18 (6), 1177-1179) and the PRS signal peptide (Sijmons et al.,1990, Bio/technology, 8:217-221). The plasmid also contains a pat geneconferring host plant phosphinothricine resistance (Wohlleben et al.,1988, Gene 70:25-37)) driven by the ubiquitin promoter/terminator fromPetroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium. Apo29 is a clone forseed-specific targeting of Apo AI to the secretory pathway.

Apo30

Apo30 (SEQ ID NO:198) is a seed-preferred clone which is constructed asper FIG. 7. As seen in FIG. 2, the Apo30 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS-pro-Apo AI (SEQ ID NO:199). Pro-Apo AI was targetedfor expression to the secretory pathway using the tobacco pathogenrelated sequence (PRS) signal peptide (Sijmons et al., 1990,Bio/technology, 8:217-221). To construct this clone forward primer 1201(SEQ ID NO:177) (5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′) adds anNcoI site to the start of pro-Apo AI. Reverse primer 1206 (SEQ IDNO:174) (5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′)adds a HindIII site after the stop codon and adds a silent mutation toremove the second XhoI site. Both primers contained extra bases on the5′ ends to facilitate RE digestion. The PCR fragment was digested withNcoI and HindIII and ligated into the plasmid SBS2090 creating plasmid4-2 (FIG. 7). Plasmid 4-2 was digested with NcoI and HindIII and the ApoAI fragment was ligated into the NcoI/HindIII sites of pSBS4011 (FIG.3D). SBS4011 contains the β-phaseolin promoter/terminator from Phaseolusvulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the Arabidopsis oleosin gene (van RooijenG. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) and the PRSsignal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). Theplasmid also contains a pat gene conferring host plant phosphinothricineresistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by theubiquitin promoter/terminator from Petroselinum crispum (Kawalleck etal., 1993, Plant. Mol. Bio., 21:673-684)) for transformation intoAgrobacterium. Apo30 is a clone for seed-specific targeting of pro-ApoAI to the secretory pathway.

Apo31

Apo31 (SEQ ID NO:200) is a seed-preferred clone which is constructed asper FIG. 8(A). As seen in FIG. 2, the Apo28 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/Apo AI (SEQ ID NO:201) fusion protein. D9ScFV/Apo AI was targeted for expression to the secretory pathway usingthe tobacco pathogen related sequence (PRS) signal peptide (Sijmons etal., 1990, Bio/technology, 8:217-221). To construct this clone forwardprimer 1200 (SEQ ID NO:188)(5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′) adds an XhoI site to thestart of pro-Apo AI. Reverse primer 1206 (SEQ ID NO: 174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. The template for these primers was the Apo33plasmid which contains the pro-form of Apo AI without internal XhoIsites and additional Met residue. The PCR fragments were cut with XhoIand ligated into the XhoI/EcoRV sites of pKS+ creating the plasmid 6-3.Plasmid 6-3 was cut with XhoI and HindIII and ligated into theXhoI/HindIII sites of binary bector SBS4055 (FIG. 9). Note SBS4055contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe tothe D9 scFV/Apo AI insert. The plasmid also contains a pat geneconferring host plant phosphinothricine resistance (Wohlleben et al.,1988, Gene 70:25-37) driven by the ubiquitin promoter/terminator fromPetroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium. Apo31 is a clone forseed-specific targeting of Apo AI to the secretory pathway andpurification with the oleosin D9 scFV antibody.

Apo32

Apo32 (SEQ ID NO:202) is a seed-preferred clone which is constructed asper FIG. 9. As seen in FIG. 2, the Apo32 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of D9 scFV/pro-Apo AI (SEQ ID NO:203). To construct thisclone forward primer 1200 (SEQ ID NO: 188)(5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′) adds an XhoI site to thestart of pro-Apo AI. Forward primer 1205 (SEQ ID NO:195)(5′-CCAAGCCCGCGCTaGAGGACCTCCG-3′) is a blunt ended primer which adds asilent mutation to remove the first XhoI site. Reverse primer 1206 (SEQID NO: 174) (5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGAGCG-3′) adds a HindIII site after the stop codon and adds a silentmutation to remove the second XhoI site. Both primers contained extrabases on the 5′ ends to facilitate restriction enzyme digestion. Thetemplate for these primers was a pKS+ based vector (Stratagene) whichcontained the entire coding sequence for human Apo AI gene. Thedouble-stranded template was removed by DpnI digestion. The PCR fragmentwas digested with XhoI and HindIII and ligated into the plasmid pKS+XhoI/HindIII sites creating plasmid P-10. P-10 was digested with XhoIand HindIII and the fragment was ligated into the XhoI/HindIII sites ofthe plasmid SBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused in frame to the D9 scFV antibody. Theplasmid also contains a pat gene conferring host plant phosphinothricineresistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by theubiquitin promoter/terminator from Petroselinum crispum (Kawalleck etal., 1993, Plant. Mol. Bio., 21:673-684) for transformation intoAgrobacterium. Apo32 is a clone targeting pro-Apo AI to the sectretorypathway fused in-frame to the oleosin antibody D9 to aid inpurification.

Apo33

Apo33 (SEQ ID NO:204) is a seed-preferred clone which is constructed asper FIG. 10. As seen in FIG. 2, the Apo33 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/met/Apo AI/ (SEQ ID NO:205) fusion protein. D9ScFV/met/Apo AI was targeted for expression to the secretory pathwayusing the tobacco pathogen related sequence (PRS) signal peptide(Sijmons et al., 1990, Bio/technology, 8:217-221). To construct thisclone forward primer 1203 (SEQ ID NO: 173)(5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′) adds an NcoI site to thestart of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The template for theseprimers was the P-10 plasmid (FIG. 9) which contains the pro-form of ApoAI without internal XhoI sites. The PCR fragment was digested with NcoIand HindIII and ligated into the NcoI/HindIII sites of pSBS2090 creatingplasmid 20-2. The plasmid was cut with NcoI and HindIII and the fragmentligated into the pSBS4010 vector BspHI/HindIII sites creating plasmid4010+20-2. The plasmid was cut with XhoI and HindIII and the fragmentwas ligated into the XhoI/HindIII sites of the binary vector SBS4055(FIG. 10). Note SBS4055 contains the β-phaseolin promoter/terminatorfrom Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.USA 80:1897-1901) controlling the expression of the PRS signal sequencefused inframe to the D9 scFV antibody. The plasmid also contains a patgene conferring host plant phosphinothricine resistance (Wohlleben etal., 1988, Gene 70:25-37) driven by the ubiquitin promoter/terminatorfrom Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,21:673-684) for transformation into Agrobacterium. Apo33 is aseed-specific clone which targets Apo AI to the secretory pathway andfused in-frame with the oleosin antibody D9 to aid in purification.

Apo34

Apo34 (SEQ ID NO:206) is a seed-preferred clone which is constructed asper FIG. 10. As seen in FIG. 2, the Apo34 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/met/pro-Apo AI/ (SEQ ID NO:207) fusionprotein. D9 ScFV/met/pro-Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). Toconstruct this clone forward primer 1201 (SEQ ID NO: 177)(5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′) adds an NcoI site to thestart of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. Both primers contained extra bases on the 5′ endsto facilitate restriction enzyme digestion. The template for theseprimers was the P-10 plasmid (FIG. 9) which contains the pro-form of ApoAI without internal XhoI sites. The PCR fragment was digested with NcoIand HindIII and ligated into the NcoI/HindIII sites of SBS2090 (FIG.3(B)) creating plasmid 19-2. The plasmid was cut with NcoI and HindIIIand the fragment ligated into the SBS4010 vector BspHI/HindIII sitescreating plasmid 4010+19-2. The plasmid was cut with XhoI and HindIIIand the fragment was ligated into the XhoI/HindIII sites of the binaryvector SBS4055 (FIG. 10). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe to the D9 scFV antibody. Theplasmid also contains a pat gene conferring host plant phosphinothricineresistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by theubiquitin promoter/terminator from Petroselinum crispum (Kawalleck etal., 1993, Plant. Mol. Bio., 21:673-684) for transformation intoAgrobacterium. Apo34 is a seed-specific clone which targets met/pro-ApoAI to the secretory pathway and fused in-frame with the oleosin antibodyD9 to aid in purification.

Apo35

Apo35 (SEQ ID NO:208) is a seed-preferred clone which is constructed asper FIG. 8. As seen in FIG. 2, the Apo35 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/Apo AI/KDEL (SEQ ID NO:209) fusion protein. D9ScFV/Apo AI was targeted for expression to the secretory pathway usingthe tobacco pathogen related sequence (PRS) signal peptide (Sijmons etal., 1990, Bio/technology, 8:217-221) and KDEL retention signal (Munroand Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide inthe ER. To construct this clone forward primer 1200 (SEQ ID NO:188)(5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′) adds an XhoI site to thestart of pro-Apo AI. Reverse primer 1208 (SEQ ID NO:210)(5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3)′ adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for these primers was the Apo33 plasmid which contains thepro-form of Apo AI without internal XhoI sites and additional Metresidue. The PCR fragments were cut with XhoI and ligated into theXhoI/EcoRV sites of pKS+ creating the plasmid 8-5. Plasmid 8-5 was cutwith XhoI and HindIII and ligated into the XhoI/HindIII sites of binarybector SBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe a D9 scFV insert. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium.Apo35 is a clone for seed-specific targeting of Apo AI to the secretorypathway with retention in the ER and purification with the oleosin D9scFV antibody.

Apo36

Apo36 (SEQ ID NO:211) is a seed-preferred clone which is constructed asper FIG. 11. As seen in FIG. 2, the Apo36 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/pro-Apo AI/KDEL (SEQ ID NO:212) fusionprotein. D9 ScFV/pro-Apo AI was targeted for expression to the secretorypathway using the tobacco pathogen related sequence (PRS) signal peptide(Sijmons et al., 1990, Bio/technology, 8:217-221) and KDEL retentionsignal (Munro and Pelham, 1987, Cell 48:899-907) is used to retain thepolypeptide in the ER. To construct this clone forward primer 1200 (SEQID NO:188) (5′-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3′) adds an XhoIsite and extra nucleotides to facilitate in-frame cloning into the klip8cleavage sequence to the start of pro-Apo AI. Reverse primer 1208 (SEQID NO:210) (5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds a KDELsequence before the stop codon and a HindIII site after the stop codon.The template for these primers was the P-10 (FIG. 9) plasmid whichcontains the pro-form of Apo AI without internal XhoI sites. The PCRfragment was cut with XhoI and HindIII and ligated into the XhoI/HindIIIsites of pKS+creating the plasmid 7-12. Plasmid 7-12 was cut with XhoIand HindIII and ligated into the XhoI/HindIII sites of the binary vectorSBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe a D9 scFV insert. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium.Apo36 is a clone for the seed-specific expression of pro-Apo AI targetedto the secretory pathway and is fused in-frame with the oleosin antibodyD9, and accumulates in the endoplasmic reticulum due to a KDEL signalpeptide.

Apo37

Apo37 (SEQ ID NO:213) is a seed-preferred clone which is constructed asper FIG. 12. As seen in FIG. 2, the Apo37 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/met/Apo AI/KDEL (SEQ ID NO:214) fusionprotein. D9 ScFV/met/Apo AI was targeted for expression to the secretorypathway using the tobacco pathogen related sequence (PRS) signal peptide(Sijmons et al., 1990, Bio/technology, 8:217-221) and KDEL retentionsignal (Munro and Pelham, 1987, Cell 48:899-907) is used to retain thepolypeptide in the ER. To construct this clone forward primer 1203 (SEQID NO:173) (5′-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3′) adds an NcoI siteto the start of mature Apo AI. Reverse primer 1208 (SEQ ID NO:210)(5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for these primers was the P-10 plasmid (FIG. 9) which containsthe pro-form of Apo AI without internal XhoI sites. The PCR fragment wasligated into the EcoRV sites of pKS+ plasmid creating plasmid 10-2. Theplasmid was cut with NcoI and HindIII and the fragment was ligated intothe SBS4010 vector BspHI/HindIII sites creating plasmid 4010+10-2. Theplasmid was cut with XhoI and HindIII and the fragment was ligated intothe XhoI/HindIII sites of binary vector SBS4055 (FIG. 9). Note SBS4055contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo37 is a clone which targetsmet/Apo AI to the secretory pathway and is fused in-frame with theoleosin antibody D9, and accumulate in the endoplasmic reticulum due toa KDEL signal peptide.

Apo38

Apo38 (SEQ ID NO:215) is a seed-preferred clone which is constructed asper FIG. 12. As seen in FIG. 2, the Apo38 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/met/pro-Apo AI/KDEL (SEQ ID NO:216) fusionprotein. D9 ScFV/met/pro-Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221) andKDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907) is usedto retain the polypeptide in the ER. To construct this clone forwardprimer 1201 (SEQ ID NO:177) (5′-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3′)adds an NcoI site to the start of mature Apo AI. Reverse primer 1208(SEQ ID NO:210) (5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds aKDEL sequence before the stop codon and a HindIII site after the stopcodon. The template for these primers was the P-10 plasmid (FIG. 9)which contains the pro-form of Apo AI without internal XhoI sites. ThePCR fragment was ligated into the EcoRV sites of pKS+ plasmid creatingplasmid 9-2. The plasmid was cut with NcoI and HindIII and the fragmentwas ligated into the SBS4010 vector BspHI/HindIII sites creating plasmid4010+9-2. The plasmid was cut with XhoI and HindIII and the fragment wasligated into the XhoI/HindIII sites of binary vector SBS4055 (FIG. 9).Note SBS4055 contains the β-phaseolin promoter/terminator from Phaseolusvulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo38 is a clone which targetsmet/pro-Apo AI to the secretory pathway and is fused in-frame with theoleosin antibody D9, and accumulate in the endoplasmic reticulum due toa KDEL signal peptide.

Apo39

Apo39 (SEQ ID NO:217) is a seed-preferred clone which is constructed asper FIG. 13. As seen in FIG. 2, the Apo39 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/Apo AI (SEQ ID NO:218) fusion protein.D9 ScFV/klip8/Apo AI was targeted for expression to the secretorypathway using the tobacco pathogen related sequence (PRS) signal peptide(Sijmons et al., 1990, Bio/technology, 8:217-221) and KDEL retentionsignal (Munro and Pelham, 1987, Cell 48:899-907) is used to retain thepolypeptide in the ER. This clone has a klip8 cleavage sequence (SEQ IDNO:143) to facilitate cleavage of the fusion protein with chymosin. Toconstruct this clone forward primer 1207 (SEQ ID NO:219)5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) sequence and adds a SalI siteto the start codon. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent (amplifies the startof the klip8) mutation to remove the second XhoI site. The template forthe PCR reaction was the plasmid Apo27 (FIG. 8(A)). The PCR product wascut with SalI and HindIII and ligated into pKS+ creating the plasmid13-1. The plasmid was cut with SalI and HindIII and ligated into theXhoI/HindIII sites of plasmid SBS4055 (FIG. 9). Note SBS4055 containsthe β-phaseolin promoter/terminator from Phaseolus vulgaris (Slightom etal., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling theexpression of the PRS signal sequence fused inframe a D9 scFV insert.The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37))driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684)) fortransformation into Agrobacterium. Apo39 is a clone for theseed-preferred expression of D9 scFV/klip8/Apo AI targeted to thesecretory pathway. Purification of Apo AI is achieved using the D9 scFVantibody which has affinity for the oleosin on the oil body. The proteincan be cleaved using chymosin which will cleave the klip8 site.

Apo40

Apo40 (SEQ ID NO:220) is a seed-preferred clone which is constructed asper FIG. 13. As seen in FIG. 2, the Apo40 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/pro-Apo AI (SEQ ID NO:221) fusionprotein. D9 ScFV/klip8/pro-Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). Thisclone has a klip8 cleavage sequence (SEQ ID NO:143) to facilitatecleavage of the fusion protein with chymosin. To construct this cloneforward primer 1207 (SEQ ID NO:219)5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) sequence and adds a SalI siteto the start codon. Reverse primer 1206 (SEQ ID NO:174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. The template for the PCR reaction was the plasmidApo27 (FIG. 8(A)). The PCR product was cut with SalI and HindIII andligated into pKS+ creating the plasmid 14-5. The plasmid was cut withSalI and HindIII and ligated into the XhoI/HindIII sites of plasmidSBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe a D9 scFV insert. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium.Apo40 is a clone for the seed-preferred expression of D9scFV/klip8/pro-Apo AI targeted to the secretory pathway. Purification ofpro-Apo AI is achieved using the D9 scFV antibody which has affinity forthe oleosin on the oil body. The protein can be cleaved using chymosinwhich will cleave the klip8 site.

Apo 41

Apo41 (SEQ ID NO:222) is a seed-preferred clone which is constructed asper FIG. 14. As seen in FIG. 2, the Apo41 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/met/Apo AI (SEQ ID NO:223) fusionprotein. D9 ScFV/klip8/met/Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). Thisclone has a klip8 cleavage sequence (SEQ ID NO:143) to facilitatecleavage of the fusion protein with chymosin. To construct this cloneforward primer 1207 (SEQ ID NO:219)(5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) amplifies the start of theklip8 sequence and adds a SalI site to the start codon. Reverse primer1206 (SEQ ID NO: 174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. The template for the PCR reaction was the plasmidApo25 (FIG. 6). The PCR product was cut with SalI and HindIII andligated into pKS+ creating the plasmid 11-1. The plasmid was cut withSalI and HindIII and ligated into the XhoI/HindIII site of binary vectorSBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe a D9 scFV insert. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684)) for transformation into Agrobacterium.Apo41 is a clone for the seed-preferred expression of D9scFV/klip8/met/Apo AI targeted to the secretory pathway. Purification ofmet/Apo AI is achieved using the D9 scFV antibody which has affinity forthe oleosin on the oil body. The protein can be cleaved using chymosinwhich will cleave the klip8 site.

Apo42

Apo42 (SEQ ID NO:224) is a seed-preferred clone which is constructed asper FIG. 14. As seen in FIG. 2, the Apo42 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/met/pro-Apo AI (SEQ ID NO:225) fusionprotein. D9 ScFV/klip8/met/pro-Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). Thisclone has a klip8 cleavage sequence (SEQ ID NO:143) to facilitatecleavage of the fusion protein with chymosin. To construct this cloneforward primer 1207 (SEQ ID NO:210)(5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) amplifies the start of theklip8 sequence and adds a SalI site to the start codon. Reverse primer1206 (SEQ ID NO: 174)(5′-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3′) adds aHindIII site after the stop codon and adds a silent mutation to removethe second XhoI site. The template for the PCR reaction was the plasmidApo26 (FIG. 7). The PCR product was cut with SalI and HindIII andligated into pKS+ creating the plasmid 12-1. The plasmid was cut withSalI and HindIII and ligated into the XhoI/HindIII site of binary vectorSBS4055 (FIG. 9). Note SBS4055 contains the β-phaseolinpromoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression ofthe PRS signal sequence fused inframe a D9 scFV insert. The plasmid alsocontains a pat gene conferring host plant phosphinothricine resistance(Wohlleben et al., 1988, Gene 70:25-37)) driven by the ubiquitinpromoter/terminator from Petroselinum crispum (Kawalleck et al., 1993,Plant. Mol. Bio., 21:673-684) for transformation into Agrobacterium.Apo42 is a clone for the seed-preferred expression of D9scFV/klip8/met/pro-Apo AI targeted to the secretory pathway.Purification of met/Apo AI is achieved using the D9 scFV antibody whichhas affinity for the oleosin on the oil body. The protein can be cleavedusing chymosin which will cleave the klip8 site.

Apo43

Apo43 (SEQ ID NO:226) is a seed-preferred clone which is constructed asper FIG. 13. As seen in FIG. 2, the Apo43 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/Apo AI/KDEL (SEQ ID NO:227) fusionprotein. D9 ScFV/klip8/Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221) andKDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907) is usedto retain the polypeptide in the ER. This clone has a klip8 cleavagesequence (SEQ ID NO:143) to facilitate cleavage of the fusion proteinwith chymosin. To construct this clone forward primer 1207 (SEQ ID NO:219) 5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) sequence and adds a SalIsite to the start codon. Reverse primer 1208 (SEQ ID NO:210)(5′-AAGCTTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for the PCR reaction was the plasmid Apo27 (FIG. 8(A)). The PCRproduct was cut with SalI and HindIII and ligated into pKS+ creating theplasmid 17-1. The plasmid was cut with SalI and HindIII and ligated intothe XhoI/HindIII sites of plasmid SBS4055 (FIG. 9). Note SBS4055contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo43 is a clone for theseed-preferred expression of D9 scFV/klip8/Apo AI/KDEL targeted to thesecretory pathway. Apo43 will accumulate in the ER due to the KDELsignal peptide. Purification of Apo AI is achieved using the D9 scFVantibody which has affinity for the oleosin on the oil body. The proteincan be cleaved using chymosin which will cleave the klip8 site.

Apo44

Apo44 (SEQ ID NO:228) is a seed-preferred clone which is constructed asper FIG. 13. As seen in FIG. 2, the Apo44 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/pro-Apo AI/KDEL (SEQ ID NO:229) fusionprotein. D9 ScFV/klip8/pro-Apo AI/KDEL was targeted for expression tothe secretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221) andKDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907) is usedto retain the polypeptide in the ER. This clone has a klip8 cleavagesequence (SEQ ID NO:143) to facilitate cleavage of the fusion proteinwith chymosin. To construct this clone forward primer 1207 (SEQ IDNO:219) 5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) sequence and adds aSalI site to the start codon. Reverse primer 1208 (SEQ ID NO:210)(5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for the PCR reaction was the plasmid Apo28 (FIG. 9). The PCRproduct was cut with SalI and HindIII and ligated into pKS+ creating theplasmid 18-2. The plasmid was cut with SalI and HindIII and ligated intothe XhoI/HindIII sites of plasmid SBS4055 (FIG. 9). Note SBS4055contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo44 is a clone for theseed-preferred expression of D9 scFV/klip8/pro-Apo AI/KDEL targeted tothe secretory pathway. Apo44 will accumulate in the ER due to the KDELsignal peptide. Purification of pro-Apo AI is achieved using the D9 scFVantibody which has affinity for the oleosin on the oil body. The proteincan be cleaved using chymosin which will cleave the klip8 site.

Apo45

Apo45 (SEQ ID NO:230) is a seed-preferred clone which is constructed asper FIG. 14. As seen in FIG. 2, the Apo45 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/met/Apo AI/KDEL (SEQ ID NO:231) fusionprotein. D9 ScFV/klip8/met/Apo AI was targeted for expression to thesecretory pathway using the tobacco pathogen related sequence (PRS)signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221) andKDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907) is usedto retain the polypeptide in the ER. This clone has a klip8 cleavagesequence (SEQ ID NO:143) to facilitate cleavage of the fusion proteinwith chymosin. To construct this clone forward primer 1207 (SEQ IDNO:219) (5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) amplifies the startof the klip8 sequence and adds a SalI site to the start codon. Reverseprimer 1208 (SEQ ID NO: 210)(5′-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3′) adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for the PCR reaction was the plasmid Apo25 (FIG. 6). The PCRproduct was cut with SalI and HindIII and ligated into pKS+ creating theplasmid 15-1. This plasmid was cut with SalI and HindIII and ligatedinto the XhoI/HindIII site of binary vector SBS4055 (FIG. 9). NoteSBS4055 contains the β-phaseolin promoter/terminator from Phaseolusvulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo45 is a clone for theseed-preferred expression of D9 scFV/klip8/met/Apo AI/KDEL targeted tothe secretory pathway. Apo45 will accumulate in the ER due to the KDELsignal peptide. Purification of Apo AI is achieved using the D9 scFVantibody which has affinity for the oleosin on the oil body. The proteincan be cleaved using chymosin which will cleave the klip8 site.

Apo46

Apo46 (SEQ ID NO:232) is a seed-preferred clone which is constructed asper FIG. 14. As seen in FIG. 2, the Apo46 clone consists of aseed-preferred promoter and terminator (phaseolin) driving theexpression of PRS/D9 scFV/klip8/met/pro-Apo AI/KDEL (SEQ ID NO:233)fusion protein. D9 ScFV/klip8/met/pro-Apo AI was targeted for expressionto the secretory pathway using the tobacco pathogen related sequence(PRS) signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221)and KDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907) isused to retain the polypeptide in the ER. This clone has a klip8cleavage sequence (SEQ ID NO:143) to facilitate cleavage of the fusionprotein with chymosin. To construct this clone forward primer 1207 (SEQID NO:219) (5′-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3′) amplifies thestart of the klip8 sequence and adds a SalI site to the start codon.Reverse primer 1208 (SEQ ID NO: 210)(5′-AAGCTTTCAtagctcatctUCTGGGTGTTGAGCTTCTTAG-3′) adds a KDEL sequencebefore the stop codon and a HindIII site after the stop codon. Thetemplate for the PCR reaction was the plasmid Apo26 (FIG. 7). The PCRproduct was cut with SalI and HindIII and ligated into pKS+ creating theplasmid 16-4. The plasmid was cut with SalI and HindIII and ligated intothe XhoI/HindIII site of binary vector SBS4055 (FIG. 9). Note SBS4055contains the β-phaseolin promoter/terminator from Phaseolus vulgaris(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)controlling the expression of the PRS signal sequence fused inframe a D9scFV insert. The plasmid also contains a pat gene conferring host plantphosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37)driven by the ubiquitin promoter/terminator from Petroselinum crispum(Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684) fortransformation into Agrobacterium. Apo46 is a clone for theseed-preferred expression of D9 scFV/klip8/met/pro-Apo AI targeted tothe secretory pathway and retained in the ER. Purification of Apo AI isachieved using the D9 scFV antibody which has affinity for the oleosinon the oil body. The protein can be cleaved using chymosin which willcleave the klip8 site.

Apo47

Apo47 (SEQ ID NO:234) is a seed-preferred clone which is constructed asper FIG. 15. As seen in FIG. 2, the Apo47 clone consists of aseed-preferred promoter and terminator (linin) driving the expression ofmaize oleosin/klip8/met/Apo AI (SEQ ID NO:235). This construct has aklip8 cleavage sequence (SEQ ID NO:143) to facilitate cleavage of thefusion protein with chymosin. To construct this clone, forward primer1226 (5′-GCAGCACCATGGCTGATCACCACCG-3′) (SEQ ID NO: 236) is used toamplify the coding sequence of maize oleosin from pSBS2377 and adds anNcoI site to the start of gene in combination with reverse primer 1227(5′-GTGGTGAAGCTTAGACCCCTGCGCC-3′) (SEQ ID NO:237) which removes the stopcodon of the gene and adds a HindIII site to assist in creating anin-frame translation fusion with klip8/met/Apo AI. The coding sequenceof klip8/met/Apo AI from Apo25 is amplified using forward primer 1228(5′-GCAGCAAAGCTTATGGCTGAGATCAC-3′) (SEQ ID NO:238) which amplifies thesequence and adds a HindIII site to the start of gene. Reverse primer1229 (5′-GTGTGGGATCCTCACTGGGTGTTG-3′) (SEQ ID NO:239) adds a BamHI siteafter the stop codon. The PCR fragments containing the maize oleosincDNA and klip8/met/Apo AI were ligated into the EcoRI sites of the Topocloning vector (Invitrogen). Plasmid MzOleo was cut with restrictionenzymes NcoI and HindIII. Plasmid klip8/met/Apo AI was cut with HindIIIand BamHI. The fragments of MzOleo and klip8/met/Apo AI were ligatedtogether into the NcoI and BamHI sites of the plasmid SBS5709 to createthe plasmid Apo47. pSBS5709 contains the linin promoter/terminator fromWO 01/16340 flanking a multiple cloning site. pSBS5709 also contains thepmi gene (Miles et al., 1984, Gene 21:41-48), encoding forphosphomannose isomerase which allows for positive selection on mannosecontaining selection media. The pmi gene is under the control of theubiquitin promoter/terminator from Petroselinum crispum (Kawalleck etal., 1993, Plant. Mol. Bio., 21:673-684)) for transformation intoAgrobacterium. Apo47 is a clone for seed-specific targeting of maizeoleosin/klip8/met/Apo AI to the oil bodies and purification using theklip8 cleavage sequence.

Example 2 Agrobacterium and Arabidopsis Transformation

Arabidopsis thaliana cv. Columbia (C24) is used for all the experiments.Seeds are planted on the surface of a soil mixture (two-thirdsRedi-earth and one-third perlite with a pH=6.7) or an Arabidopsis soilmixture supplied by Lehle Seeds (perlite, vermiculite, peat,terra-green, with a pH=5.5) in 4 inch pots. The seedlings are allowed togrow to a rosette stage of 6-8 leaves to a diameter of approximately 2.5cm. These seedlings are transplanted into 4 inch pots containing theabove soil mixture, covered with window screen material which has five 1cm diameter holes cut into the mesh; one in each of the corners, and onein the center. The pots are placed inside a dome at 4° C. for four daysfor a cold treatment and subsequently moved to 24° C. growth room withconstant light at about 150 μE and 50% relative humidity. The plants areirrigated at 2-3 day interval and fertilized weekly with 1% of Peters20-20-20. Each pot contains five plants. When plants reach about 2 cm inheight, the primary bolts are cut to encourage the growth of secondaryand tertiary bolts. 4 to 5 days after cutting the primary bolts, theplants are ready to be infected with Agrobacterium. The plasmid wastransformed into electrocompetent Agrobacterium EHA101. The pots withArabidopsis plants are inverted and infected with 500 ml of are-suspension an overnight Agrobacterium culture containing the planttransformation vector of interest for 20 seconds. It is critical thatthe Agrobacterium culture contains 5% sucrose and 0.05% of thesurfactant Silwet L-77 (Lehle Seeds). The pots are subsequently coveredwith a trans-parent plastic dome for 24 hours to maintain higherhumidity. The plants are allowed to grow to maturity and seeds(untransformed and transformed) are harvested. For selection oftransgenic lines, the putative transformed seeds are sterilized in aquick wash of 70% ethanol, then 20% commercial bleach for 15 min andthen rinsed at least four times with ddH₂O. About 1000 sterilized seedsare mixed with 0.6% top agar and evenly spread on a half strength MSplate (Murashige and Skoog, 1962, Physiologia Plantarum 15: 473-497)containing 0.5% sucrose and 80 μM of the herbicide phosphinothricin(PPT) DL. The plates are then placed in a growth room with light regime8 hr dark and 16 hr light at 24° C. After 7 to 10 days, putativetransgenic seedlings are green and growing whereas untransformedseedlings are bleached. After the establishment of roots the putativetransgenic seedlings are individually transferred to pots (theindividually plants are irrigated in 3 day interval and fertilized with1% Peters 20-20-20 in 5 day interval) and allowed to grow to maturity.The pots are covered with a transparent plastic dome for three days toprotect the sensitive seedlings. After 7 days the seedlings are coveredwith a seed collector from Lehle Seeds to prevent seed loss due toscattering. Seeds from these transgenic plants are harvestedindividually and ready for analysis.

Total Leaf Extract Preparation

An Arabidopsis leaf was frozen with liquid nitrogen and ground in a 1.5ml microfuge tube using a drill. 200-250 μl of 0.5M Tris-HCl, pH 7.5 wasadded and the sample put on ice. 20% SDS was added to a finalconcentration of 2%. The sample was boiled for 5 minutes and the extractwas spun in a microfuge at maximum speed for 5 minutes. The liquid wasremoved to another microfuge tube and stored at −20° C. Soluble proteinswere quantified using the BCA Protein assay (Pierce) and analyzed on a15% SDS-PAGE followed by Western blotting. An anti-Apo AI or anti-GFPrabbit antiserum was used as the primary antibody; and anti-rabbit-IgG[H+L]-AP conjugate (Bio-Rad) was used as the secondary antibody.

Total Seed Extract Preparation

Approximately 40 Arabidopsis seeds (T2 seed) were ground in 50 uL buffer(50 mM Tris pH 7.5) in microfuge tube using Stir-Pak laboratory mixer.20% SDS was subsequently added to the sample to a final concentration of2% and the sample was boiled for 5 minutes and centrifuged at maximumspeed for 5 minutes. For loading onto an SDS-PAGE gel, SDS-PAGE 2Xloading buffer (100 mM Tris pH 6.8, 20% glycerol, 4% SDS, 2 mg/mLbromophenol blue, 200 mM DTT) and 1M DTT were added to sample, boiledfor 5 minutes and centrifuged at maximum speed for 2 minutes.

Example 3 Western Blot Analysis for Apolipoprotein ExpressionConstitutive Expression

Apo17

As seen in Example 1, Apo17 (SEQ ID NO:27) is a fusion protein betweenmature Apo AI and GFP. An ubiquitin promoter and terminator are used forconstitutive expression of the construct. Western blot analysis (FIG.16(A)) using a polyclonal Apo AI antibody detected very low amounts(less than 0.1% of total leaf protein) the Apo-AI/GFP fusion protein ina total leaf extract at a molecular weight of approximately 55 kDa in 9of the 12 clones. However substantial expression (at least 1% of totalseed protein) of the Apo17 fusion protein was detected in a total seedextract (FIG. 16(B)) at approximately 55 kDa in 11 out of 12 clonestested.

Apo18a

As seen in Example 1, Apo18 (SEQ ID NO:29) is a fusion protein betweenpro-Apo AI and GFP. An ubiquitin promoter and terminator are used forexpression of the construct. Western blot analysis (FIG. 17(A)) using apolyclonal Apo AI antibody detected very low amounts (less than 0.1% oftotal leaf protein) of the pro-Apo AI/GFP fusion protein in a total leafextract at a molecular weight of approximately 56 kDa in 3 of the 12clones tested. However substantial expression (at least 1% of total seedprotein) of the Apo18 fusion protein was detected in a total seedextract (FIG. 17(B)) at approximately 56 kDa in all clones tested.

Apo19

As seen in Example 1, Apo19 (SEQ ID NO:31) is a fusion protein betweenApo AI and GFP. An ubiquitin promoter and terminator are used forexpression of the construct and the PRS signal peptide is used for thetargeted expression of the fusion protein to the ER. Western blotanalysis (FIG. 18(A)) using a polyclonal Apo AI antibody detected verylow levels (less than 0.1% of total leaf protein) of the Apo AI/GFPfusion protein in a total leaf extract at a molecular weight ofapproximately 58 kDa in 10 of the 12 clones tested. However substantialexpression (at least 1% of total seed protein) of the Apo19 fusionprotein was detected in a total seed extract (FIG. 18(B)) atapproximately 58 kDa in 17 of the 18 clones tested.

Apo20

As seen in Example 1, Apo20 (SEQ ID NO:35) is a fusion protein betweenpro-Apo AI and GFP. An ubiquitin promoter and terminator are used forexpression of the construct and the PRS signal peptide is used for thetargeted expression of the fusion protein to the ER. Western blotanalysis (FIG. 19(A)) using a polyclonal Apo AI antibody detected verylow levels (less than 0.1% of total leaf protein) of the pro-Apo AI/GFPfusion protein in a total leaf extract at a molecular weight ofapproximately 59 kDa in 3 of the 12 clones tested. However substantialexpression (at least 1% of total seed protein) of the Apo20 fusionprotein was detected in a total seed extract (FIG. 19(B)) atapproximately 59 kDa in 16 of the 18 clones tested.

Seed-Specific Expression

Apo10

As seen in Example 1, Apo10 (SEQ ID NO:10) is a fusion protein betweenmature Apo AI and GFP. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.20(A)) using a polyclonal GFP antibody detected the Apo AI/GFP fusionprotein in a total seed extract at a molecular weight of approximately55 kDa in 7 of the 10 clones tested.

Apo11

As seen in Example 1, Apo11 (SEQ ID NO:16) is a fusion protein betweenpro-Apo AI and GFP. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.20(B)) using a polyclonal GFP antibody detected the pro-Apo AI/GFPfusion protein in a total seed extract at a molecular weight ofapproximately 56 kDa in 10 of the 14 clones tested.

Apo12

As seen in Example 1, Apo12 (SEQ ID NO:19) is a fusion protein betweenoleosin, mature Apo AI and GFP. A phaseolin promoter and terminator areused for seed-specific expression of the construct. Western blotanalysis (FIG. 21(A)) using a polyclonal GFP antibody detected the ApoAI/GFP fusion protein in a total seed extract at a molecular weight ofapproximately 74 kDa in all of the 17 clones tested.

Apo13

As seen in Example 1, Apo13 (SEQ ID NO:21) is a fusion protein betweenoleosin, pro-Apo AI and GFP. A phaseolin promoter and terminator areused for seed-specific expression of the construct. Western blotanalysis (FIG. 21(B)) using a polyclonal GFP antibody detected theoleosin/pro-Apo AI/GFP fusion protein in a total seed extract at amolecular weight of approximately 75 kDa in all 18 of the clones tested.

Apo15

As seen in Example 1, Apo15 (SEQ ID NO:23) is a fusion protein betweenmature Apo AI and GFP. A phaseolin promoter and terminator are used forseed-specific expression of the construct and the PRS signal peptide isused for the targeted expression of the fusion protein to the secretorysystem. Western blot analysis (FIG. 22(A)) using a polyclonal GFPantibody detected the Apo AI/GFP fusion protein in a total seed extractat a molecular weight of approximately 58 kDa in 9 of the 10 clonestested.

Apo16

As seen in Example 1, Apo16 (SEQ ID NO:25) is a fusion protein betweenpro-Apo AI and GFP. A phaseolin promoter and terminator are used forseed-specific expression of the construct and the PRS signal peptide isused for the targeted expression of the fusion protein to the secretorypathway. Western blot analysis (FIG. 22(B)) using a polyclonal GFPantibody detected the pro-Apo AI/GFP fusion protein in a total seedextract at a molecular weight of approximately 59 kDa in 10 of the 13clones tested.

Apo21

As seen in Example 1, Apo21 (SEQ ID NO:37) is Apo AI. A phaseolinpromoter and terminator are used for seed-specific expression of theconstruct. Western blot analysis (FIG. 23(A)) using a polyclonal Apo AIantibody was used to detect the Apo AI protein in a total seed extract.The expected molecular weight was approximately 28 kDa. In the 12 clonestested, a number of different proteins were detected with the Apo AIantibody which ranged from approximately 25 kDa to upwards of 55 kDa. Itshould be noted that the expression of Apo AI was detrimental to thehealth of the plants (i.e. stunted siliques and the absence of seeds).

Apo22

As seen in Example 1, Apo22 (SEQ ID NO:41) is pro-Apo AI. A phaseolinpromoter and terminator are used for seed-specific expression of theconstruct. Western blot analysis (FIG. 23(B)) using a polyclonal Apo AIantibody was used to detect the Apo AI protein in a total seed extract.The expected molecular weight was approximately 29 kDa. In the 6 clonestested, a number of different proteins were detected with the Apo AIantibody which ranged from approximately 25 kDa to upwards of 55 kDa.Clone 22-3 has a protein of the appropriate molecular weight. It shouldbe noted that the expression of pro-Apo AI was somewhat detrimental tothe health of the plants with an intermediate phenotype when compared tothe health of plants expressing constructs Apo21, Apo29 and Apo30 versusApo23.

Apo23

As seen in Example 1, Apo23 (SEQ ID NO:44) is a fusion protein betweenoleosin and Apo AI. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.24(A)) using a polyclonal Apo AI antibody detected the oleosin/Apo AIfusion protein in a total seed extract at a molecular weight ofapproximately 47 kDa in 4 of the 5 clones tested.

Apo24

As seen in Example 1, Apo24 (SEQ ID NO:46) is a fusion protein betweenoleosin and pro-Apo AI. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.24(B)) using a polyclonal Apo AI antibody detected the oleosin/Apo AIfusion protein in a total seed extract at a molecular weight ofapproximately 48 kDa in all 7 clones tested.

Apo25

As seen in Example 1, Apo25 (SEQ ID NO:48) is a fusion protein betweenoleosin and Apo AI(+Met) with a klip8 cleavage sequence separating thetwo components. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.25(A)) using a polyclonal Apo AI antibody detected the oleosin-klip8-ApoAI(+Met) fusion protein in a total seed extract at a molecular weight ofapproximately 51 kDa in all 4 of the clones tested.

Apo26

As seen in Example 1, Apo26 (SEQ ID NO:51) is a fusion protein betweenoleosin and pro-Apo AI(+Met) with a klip8 cleavage sequence separatingthe two components. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.25(B)) using a polyclonal Apo AI antibody detected theoleosin-klip8-proApo AI(+Met) fusion protein in a total seed extract ata molecular weight of approximately 52 kDa in all 11 of the clonestested.

Apo28

As seen in Example 1, Apo28 (SEQ ID NO:60) is a fusion protein betweenoleosin and pro-Apo AI with a klip8 cleavage sequence separating the twocomponents. A phaseolin promoter and terminator are used forseed-specific expression of the construct. Western blot analysis (FIG.26(A)) using a polyclonal Apo AI antibody detected theoleosin-klip8-pro-Apo AI fusion protein in a total seed extract at amolecular weight of approximately 52 kDa in all 7 of the clones tested.

Apo29

As seen in Example 1, Apo29 (SEQ ID NO:63) is Apo AI. A phaseolinpromoter and terminator are used for seed-specific expression of theconstruct and the PRS signal peptide is used for the targeted expressionof the protein to the secretory pathway. Western blot analysis (FIG.26(B)) using a polyclonal Apo AI antibody was used to detect the Apo AIprotein in a total seed extract. The expected molecular weight wasapproximately 31 kDa. In the 10 clones tested, only 1 clone had aprotein detected but the molecular weight was in the range of 37 kDa. Itshould be noted that the expression of Apo AI was detrimental to thehealth of the plants (i.e. stunted siliques and the absence of seeds).

Apo30

As seen in Example 1, Apo30 (SEQ ID NO:65) is pro-Apo AI. A phaseolinpromoter and terminator are used for seed-specific expression of theconstruct and the PRS signal peptide is used for the targeted expressionof the fusion protein to the secretory pathway. Western blot analysis(FIG. 27) using a polyclonal Apo AI antibody was used to detect the ApoAI protein in a total seed extract. The expected molecular weight wasapproximately 32 kDa. In the 13 clones tested, a number of differentproteins were detected with the Apo AI antibody which ranged fromapproximately 25 kDa to upwards of 55 kDa. It should be noted that theexpression of pro-Apo AI was detrimental to the health of the plants(i.e. stunted siliques and the absence of seeds).

Apo32

As seen in Example 1, Apo32 (SEQ ID NO:69) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI. A phaseolin promoter and terminatorare used for seed-specific expression of the construct and the PRSsignal peptide is used for the targeted expression of the fusion proteinto the secretory pathway. Western blot analysis (FIG. 28(A)) using apolyclonal Apo AI antibody detected the D9 scFV-pro-Apo AI fusionprotein in a total seed extract at a molecular weight of approximately59 kDa in all 5 of the clones tested.

Apo33

As seen in Example 1, Apo33 (SEQ ID NO:71) is a fusion protein betweenthe D9 scFV antibody and Apo AI(+met). A phaseolin promoter andterminator are used for seed-specific expression of the construct andthe PRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway. Western blot analysis (FIG. 28(B))using a polyclonal Apo AI antibody detected the D9 scFV-Apo AI(+met)fusion protein in a total seed extract at a molecular weight ofapproximately 59 kDa in 1 of the 8 clones tested.

Apo34

As seen in Example 1, Apo34 (SEQ ID NO:73) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI(+met). A phaseolin promoter andterminator are used for seed-specific expression of the construct andthe PRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway. Western blot analysis (FIG. 29) usinga polyclonal Apo AI antibody detected the D9 scFV-pro-Apo AI(+met)fusion protein in a total seed extract at a molecular weight ofapproximately 59 kDa in 6 of the 7 clones tested.

Apo36

As seen in Example 1, Apo36 (SEQ ID NO:78) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI. A phaseolin promoter and terminatorare used for seed-specific expression of the construct, the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway and KDEL retention signal is used to retain thepolypeptide in the ER. Western blot analysis (FIG. 30(A)) using apolyclonal Apo AI antibody detected the D9 scFV-pro-Apo AI-KDEL fusionprotein in a total seed extract at a molecular weight of approximately60 kDa in all 13 of the clones tested.

Apo37

As seen in Example 1, Apo37 (SEQ ID NO:80) is a fusion protein betweenthe D9 scFV antibody and Apo AI(+met). A phaseolin promoter andterminator are used for seed-specific expression of the construct, thePRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway and KDEL retention signal is used toretain the polypeptide in the ER. Western blot analysis (FIG. 30(B))using a polyclonal Apo AI antibody detected the D9 scFV-ApoAI(+met)-KDEL fusion protein in a total seed extract at a molecularweight of approximately 59 kDa in all 15 of the clones tested.

Apo38

As seen in Example 1, Apo38 (SEQ ID NO:82) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI(+met). A phaseolin promoter andterminator are used for seed-specific expression of the construct, thePRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway and KDEL retention signal is used toretain the polypeptide in the ER. Western blot analysis (FIG. 31A) usinga polyclonal Apo AI antibody detected the D9 scFV-pro-Apo AI(+met)-KDELfusion protein in a total seed extract at a molecular weight ofapproximately 60 kDa in 9 of the 11 clones tested.

Apo39

As seen in Example 1, Apo39 (SEQ ID NO:83) is a fusion protein betweenthe D9 scFV antibody, KLIP8 and Apo AI. A phaseolin promoter andterminator are used for seed-specific expression of the construct, thePRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway and KLIP8 is used as a cleavablelinker. Western blot analysis (FIG. 31B) using a polyclonal Apo AIantibody detected the D9 scFV-KLIP8-Apo AI fusion protein in a totalseed extract at a molecular weight of approximately 55 kDa in all 12 ofthe clones tested.

Apo40

As seen in Example 1, Apo40 (SEQ ID NO:87) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI with a klip8 cleavage sequenceseparating the two components. A phaseolin promoter and terminator areused for seed-specific expression of the construct and the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway. Western blot analysis (FIG. 32(A)) using a polyclonalApo AI antibody detected the D9 scFV-klip8-pro-Apo AI fusion protein ina total seed extract at a molecular weight of approximately 63 kDa in 12of the 13 clones tested.

Apo41

As seen in Example 1, Apo41 (SEQ ID NO:89) is a fusion protein betweenthe D9 scFV antibody and Apo AI(+met) with a klip8 cleavage sequenceseparating the two components. A phaseolin promoter and terminator areused for seed-specific expression of the construct and the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway. Western blot analysis (FIG. 32(B)) using a polyclonalApo AI antibody detected the D9 scFV-klip8-Apo AI(+met) fusion proteinin a total seed extract at a molecular weight of approximately 63 kDa in8 of the 9 clones tested.

Apo42

As seen in Example 1, Apo42 (SEQ ID NO:91) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI(+met) with a klip8 cleavage sequenceseparating the two components. A phaseolin promoter and terminator areused for seed-specific expression of the construct and the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway. Western blot analysis (FIG. 33(A)) using a polyclonalApo AI antibody detected the D9 scFV-klip8-pro-Apo AI(+met) fusionprotein in a total seed extract at a molecular weight of approximately64 kDa in all of the 13 clones tested.

Apo44

As seen in Example 1, Apo44 (SEQ ID NO:95) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI. A phaseolin promoter and terminatorare used for seed-specific expression of the construct, the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway and KDEL retention signal is used to retain thepolypeptide in the ER. Western blot analysis (FIG. 34(A)) using apolyclonal Apo AI antibody detected the D9 scFV-pro-Apo AI-KDEL fusionprotein in a total seed extract at a molecular weight of approximately64 kDa in 4 of the 15 clones tested.

Apo45

As seen in Example 1, Apo45 (SEQ ID NO:97) is a fusion protein betweenthe D9 scFV antibody and Apo AI(+met). A phaseolin promoter andterminator are used for seed-specific expression of the construct, thePRS signal peptide is used for the targeted expression of the fusionprotein to the secretory pathway and KDEL retention signal is used toretain the polypeptide in the ER. Western blot analysis (FIG. 34(B))using a polyclonal Apo AI antibody detected the D9 scFV-ApoAI(+met)-KDEL fusion protein in a total seed extract at a molecularweight of approximately 63 kDa in all 12 of the clones tested.

Apo46

As seen in Example 1, Apo46 (SEQ ID NO:99) is a fusion protein betweenthe D9 scFV antibody and pro-Apo AI(+met) with a klip8 cleavage sequenceseparating the two components. A phaseolin promoter and terminator areused for seed-specific expression of the construct, the PRS signalpeptide is used for the targeted expression of the fusion protein to thesecretory pathway and KDEL retention signal is used to retain thepolypeptide in the ER. Western blot analysis (FIG. 35(A)) using apolyclonal Apo AI antibody detected the D9 scFV-klip8-pro-ApoAI(+met)-KDEL fusion protein in a total seed extract at a molecularweight of approximately 64 kDa in 10 of the 11 clones tested.

Example 4 Western Blot Analysis for Apolipoprotein Localization

The isolation of oil bodies was performed as previously described (vanRooijen & Moloney, 1995) with the following modifications. Briefly, 250mg of dry mature seeds were surface sterilized with 70% ethanol, rinsedtwice with sterile water and once with a phosphate buffer (100 mMphosphate buffer pH 8 with 0.5M NaCl). After washing, the seeds wereresuspended in phosphate buffer for analytical analysis and then groundusing a sterilized mortar and pestle. After grinding, the sample wastransferred to a centrifuge bottle and centrifuged for 15 min at 10,000g at RT. After centrifugation, the fat pad containing the oil bodies wasremoved from the aqueous phase (AQ) and transferred to a 1.5 mLmicrofuge tube. The oil bodies were resuspended in a low stringencyphosphate buffer (100 mM phosphate buffer pH 8 with 0.5M NaCl). Thesample was centrifuged for 15 minutes at 10,000 g at 4° C. and theundertatant removed. The undernatant (PW) and phosphate washed oilbodies (PO) were tested for the presence of the apolipoprotein. The oilbodies were subsequently resuspended in a high stringency urea buffer(8M Urea in 100 mM Na-Carbonate buffer pH 8). The sample was centrifugedfor 15 min at 10,000 g at 4° C. and the undernatant removed. Theundernatant (UW) and urea washed oil bodies (UO) were tested for thepresence of the apolipoprotein. Note that oil bodies are treated withlow and high stringency washes in order to remove proteins whichassociate with the oil bodies. Oleosin resists high stringency washesand remains with the oil body fraction. The microsomal fraction (ER) wasobtained by grinding approximately 250 mg of dry seeds in 4 mL ofmicrosome grinding buffer (0.5M Sucrose, 0.2M Hepes-NaOH buffer, pH7.4), into a slurry with a mortar and pestle. The slurry was centrifugedat 10,000 g for 30 min at 4° C. The supernatant was transferred into newtubes, and recentrifuged at 10,000 g for another 30 min at 4° C. tocompletely remove oil bodies. The supernatant was then centrifuged for 2hrs at 100,000 g at 4° C. using an ultracentrifuge with a swingingbucket rotor. The pellet was washed with microsome grinding buffer,quickly centrifuged for 5 min for 10,000 g and resuspended in 10-15 uLof microsome grinding buffer and stored at −20° C.

Untargeted Constructs

As seen in FIG. 36A, pro-(Apo11) and mature (Apo10) Apo AI-GFP fusions,without additional targeting signals, were examined in the differentfractions. Apo10 shows that while the mature Apo AI-GFP fusion proteinis detected in all cellular fractions, there appears to be more proteinaccumulation with the oil bodies washed with phosphate buffer and theurea wash fraction. This suggests that while Apo10 does possess someaffinity to oil bodies, a high stringency wash is sufficient to removeit from the surface of the oil bodies. Apo11 shows that the presence ofthe native pro-sequence of Apo AI targets the Apo AI-GFP fusion proteinto the oil body fraction to a greater extent than when the pro-peptideis missing, and that the protein remains bound to the oil-bodies evenwhen high stringency washes are used. It appears that the pro-Apo AIpeptide acts as an ‘anchoring’ sequence to maintain pro-Apo AI on thesurface on the oil bodies. Multiple lower molecular weight bands can bedetected in these fractions, which may be degradation products.

Oil Body Targeted Constructs

This pattern of tight association of Apo AI-GFP fusion protein with oilbodies can also be seen in the oil body targeted constructs (FIG. 36B).Pro-(Apo13) and mature (Apo12) Apo AI-GFP fusions are fused in-frame tothe C-terminus of oleosin, which serves as a targeting signal to oilbodies. As seen previously for the untargeted pro-Apo AI-GFP fusion, theprotein is predominantly associated with the phosphate and urea-washedoil body fractions. Multiple lower molecular weight bands are alsodetected in these fractions, indicating that there is something uniqueto the oil body associated fractions which leads to an accumulation ofpossible degradation products.

Secretory System Targeted Constructs

The PRS signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221)which targets proteins to the secretory pathway was added as an in-frametranslational fusion to the pro-(Apo16) and mature (Apo15) forms of ApoAI-GFP (FIG. 37). Normally, this results in the secretion of the fusionprotein into the extracellular space (apoplast) of plant cells. However,when the cellular fractions of the two constructs were examined, the ApoAI-GFP fusion protein was detected in all of the fractions, with moreprotein being detected in the oil body fractions. A band the size of therecombinant fusion protein can also be observed in the oil bodyfractions when the Ponceau-S stained immunoblot is examined (FIG. 37,upper panel). The presence of the native pro-sequence of Apo AI did notappear to change the secreted fusion protein's association with aspecific cellular fraction. The plant presequence recognition signal(PRS) peptide appears to interfere with the ‘anchoring’ characteristicof the pro-Apo AI peptide.

Constitutive Expression of Apo AI-GFP in the Leaves

Leaf tissue that constitutively expressed the untargeted and secretedpro- and mature forms of Apo AI-GFP was examined to determine if the ApoAI-GFP fusion protein could accumulate in non-oil producing tissues.Leaf tissues were homogenized (as described in example 2) from thetransgenic plants lines constitutively expressing the untargetedpro-(Apo18) and mature (Apo17) and the secreted pro-(Apo20) and mature(Apo19) forms of Apo AI-GFP and used for immunoblotting. Leaf materialwas also sampled from wild-type (c24) plants for a negative control, andfrom UR2 (ubiquitin-driven oleosin-GFP) plants and UG-14(ubiquitin-driven GFP) plants for positive controls for leaf expression.Similar amounts of protein were separated by SDS-PAGE and the Apo AI-GFPfusion protein was detected by immunoblotting with an anti-GFP antibody(FIG. 38 lower panel).

Faint bands were detected in the non-transformed line, whereas the boththe ubiquitin-driven GFP (UG-14) and oleosin-GFP (UR2) fusion arereadily detected on the immunoblot by the anti-GFP antibody. However,while a similar amount of leaf protein was loaded on the immunoblot ascan be seen by Ponceau-S staining of the membrane (upper panel, FIG.38), no bands can be detected for any of the Apo AI-GFP fusion proteinconstructs. The faint band detected in the background of the immunoblotmay be due to cross-reactivity with Rubisco, the most prevalent proteinfound in leaf tissue (Spreitzer R J & Salvucci M E (2002) Rubisco:structure, regulatory interactions, and possibilities for a betterenzyme. Annu Rev Plant Biol 53: 449-475.), which can be seen in thePonceau-S stained panel of the immunoblot by size (55 kDa) according tothe molecular weight markers.

Seed Specific Expression of Untargeted and Secreted Apo AI in Seeds

One transgenic line was selected from each of the T, generation Apo AItransgenic plants that showed some accumulation of either pro- or matureApoAI protein in total seed extracts. Of the plant lines that wereputative transgenics, only 1 line for the following showed any proteinexpression: Apo21 (untargeted mature Apo AI), Apo22 (untargeted pro-ApoAI), and Apo29 (secreted mature Apo AI); two lines were found for Apo30(secreted pro-Apo AI). All transgenic plant lines that contained theconstructs Apo23 and Apo24 appeared to express and accumulate theoleosin-Apo AI fusion protein. Similar amounts of protein were separatedby SDS-PAGE and the Apo AI protein was detected by immunoblotting withan anti-Apo AI antibody (FIG. 39). A negative control was included forthe immunoblot by preparing a similar protein extract from anon-transformed plant (c24). The correct size of band for Apo AIexpressed alone (expected molecular weight of 28.3 kDa for mature Apo AIand 29.3 kDa for pro-Apo AI) was only observed in line Apo22-3(untargeted pro-Apo AI). The predominant band detected in Apo21-11(untargeted mature Apo AI) is the incorrect size, and while only minorbands are detected in Apo29-11 (secreted mature Apo AI) and Apo30-14(secreted proApo AI) none are the correct size. However, for both of theoleosin fusions, Apo23-11 (oleosin-mature Apo AI) and Apo24-6(oleosin-pro-Apo AI), a significant amount of Apo AI fusion protein isdetected by anti-Apo AI antibody and can also be detected in thePonceau-S stained immunoblot (upper panel).

Subcellular Localization of Untargeted Apo AI (Apo22) T3 Seeds

The seed-specific expression of the pro-form of the untargeted Apo AIprotein (Apo22) and its association with a specific cellular fractionwas examined in mature seeds (FIG. 40). Seeds were homogenized andtreated as described above, and the cellular fractions were subjected toimmunoblotting with an antibody against Apo AI. Apo22-3 shows that thepresence of the native pro-sequence of Apo AI targets the Apo AI proteinto the oil body fraction, and that the protein requires high stringencywashes to remove the protein from the oil bodies. Some protein can alsobe detected in the aqueous phase indicating that not all the pro-Apo AIprotein is associated with the oil bodies. Multiple lower molecularweight bands can be detected in these fractions, which may bedegradation products.

Example 5 Confocal Microscopy for Apolipoprotein Localization

Immature embryos were dissected out of Apo AI-GFP siliques under sterilewater into a Petri dish using forceps and a dissecting microscope.Embryos were removed from the seed coat by gentle pressure on theimmature seeds with a glass microscope slide cover. Embryos weretransferred by pipette into a 1.5 mL microfuge tube with water added toa final volume of 1 mL. Nile Red (Molecular Probes) was used at a finalconcentration of 1 μg/μL. Embryos in diluted Nile Red were left toincubate for 15 min in darkness at room temperature. Embryos were rinsed3 times in sterile water and mounted in water on glass slides formicroscopy. Leaf epidermal cells were prepared by simply cutting smallportions of leaves (0.5 cm²) with a scalpel and mounting them in wateron microscope slides with cover slips. The leaf sections were placed sothat the lower epidermis faced upwards (less interference withautofluorescent chloroplasts).

All GFP-dependent fluorescence was analyzed from Arabidopsis embryos andleaf epidermal cells mounted in water for microscopic observations andexamined with a Zeiss LSM 510 laser scanning confocal microscope(Edmonton, AB). For simultaneous detection of GFP and Nile Red aline-sequential single-tracking mode with the AOTF-controlled excitationwith 488 nm and 543 nm light was set at 20% and 100% respectively. APlan-Appochromat 63x/1.4 Oil DIC objective was used with a 5× scan zoom.The pinhole was optimized for Channel 2 (green) at 94 μm and for Channel1 (red) at 106 μm.

The resulting micrographs can be seen in FIGS. 42 to 45. Colocalization(indicated in yellow) between untargeted pro-Apo AI-GFP fusions (Apo11)and Nile red stained oil bodies, is evident in FIG. 41 (D-F). There isalso colocalization (indicated in yellow) between Apo12, mature Apo AIfused to GFP targeted to oil bodies using oleosin, (G-I of FIG. 41) andApo13, pro-Apo AI fused to GFP targeted to oil bodies using oleosin (A-Cof FIG. 42). Colocalization (indicated in yellow) between untargetedpro-Apo AI-GFP fusions (Apo18) and Nile red stained oil bodies isevident in (D-F of FIG. 43). No colocalization between Apo AI-GFP fusionprotein (Apo19) and oil bodies is observed (G-H of FIG. 43). In FIG. 44,no colocalization is evident in the leaves. In conclusion, in theabsence of an oil body target (i.e. oleosin), co-localization of Apo AIis observed only in the embryos (in the presence of neutral lipid) andonly when the pro-peptide of Apo AI is expressed in the cytoplasm (i.e.not when secreted).

Example 6 Cleavage and HPLC Analysis of Apo 25, 26 and 28 ExpressingArabidopsis Seed Cleavage of Apo25, Apo26 and Apo28 Recombinant Protein

The isolation of oil bodies was performed as previously described (vanRooijen & Moloney, 1995) with the following modifications. Briefly, 250mg of dry mature seeds were surface sterilized with 70% ethanol, rinsedtwice with sterile water and once with a phosphate buffer (100 mMphosphate buffer pH 8 with 0.5M NaCl). After washing, the seeds wereresuspended in phosphate buffer for analytical analysis and then groundusing a sterilized mortar and pestle. After grinding, the sample wastransferred to a centrifuge bottle and centrifuged for 15 min at 10,000g at RT. After centrifugation, the fat pad containing the oil bodies wastransferred to a 1.5 mL microfuge tube and resuspended in a urea buffer(8M Urea in 100 mM Na-Carbonate buffer pH 8). The sample was centrifugedfor 15 min at 10,000 g at 4° C. and the undernatant removed. The fat padwas resuspended in sterile ddH₂O, centrifuged for 15 min at 10,000 g at4° C. and the undernatant removed. The oil bodies were resuspended in 50μL of sterile ddH₂O and stored in the dark at 4° C. The cleavagereaction was performed in a 20 pit reaction volume containing 100 mMphosphate buffer pH 4.5, with a final ratio of 1:100 protease to oilbody protein, at 37° C. for 2 hrs. A sample reaction would be asfollows: 20 μg of purified Apo25, Apo26 or Apo28 was combined with 2 μL1M phosphate buffer pH 4.5 (final concentration 100 mM), 2 μL, chymosin(0.1 _g/_l) with sterile ddH₂O to bring up to final volume of 20 μL.After 2 hrs, the cleavage reaction was centrifuged for 15 min, and theundernatent was removed from the fat pad and each phase was analyzed forrecombinant protein.

Purification of Apo25, Apo26 and Apo28 by Reverse Phase Chromatography

Approximately, 1000 μg of Apo25, Apo26 or Apo28 was cleaved by chymosinfor 2 hrs at 37° C. After the cleavage reaction was completed, thereaction was centrifuged for 15 min at 10,000 g at 4° C. and theundernatent was recovered. The fat pad was resupsended in a urea buffer(8M Urea in 0.1 mM Na-Carbonate buffer pH 8), and recentrifuged for 15min. The undernatent or wash was recovered, and the washes were repeatedfor an additional three times, with the undernatent being recovered eachtime and pooled into a 15 mL Falcon tube. After the urea washes werecompleted, the washes were aliquoted into 1.5 mL microfuge tubes, andcentrifuged for 15 minutes to remove any contaminating oil body residue.The undernatents were recovered and filtered into a new 15 mL Falcontube using a 0.2 micron filter. A VYDAC 214TP54 C4 silica 5 micron(Grace Vydac, Anaheim, Calif.) reverse-phase chromatography column(0.24×25 cm) was equilibrated in buffer A (10% acetonitrile and 0.1%trifluoroacetic acid) at a flow-rate of 2 mL/min. The pooledchymosin-cleaved Apo25, Apo26 or Apo28 urea undernatent was loaded onthe column. A linear gradient was applied to the column 0 to 60% bufferB (95% acetonitrile, 0.1% trifluoroacetic acid) for the elution of ApoAI. Apo AI (US Biological, Catalogue number A2299-10) was used as astandard for comparising the cleavage products from Apo25, Apo26 andApo28. Fractions 19.6′ to 20.8′ (0.2′=0.4 mL each) were collected.Comparing the relative intensities of the DAD traces at 214, 254, 280 &326 nm indicates that the material eluting in the 19.5-21.0′ zone mostlikely represents the Apo AI polypeptide (FIG. 45A). These peaks alsoincreased in intensity compared to a previous injection of 0.020 mL ofthe same sample. To purify the cleavage product from Apo25(oleosin-klip8-Apo AI(met+)), chymosin treated fractions were collectedfrom 7 to 25′ @ 1 mL each. The major polypeptide peak is at 20.5° (FIG.45B), which is just 0.2′ later than the suspected hApo AI standard. Topurify the cleavage product from Apo26 (oleosin-klip8-pro-Apo AI(met+)),fractions were collected from 7 to 25′ at 1 mL each. The majorpolypeptide peak is at 18′ (FIG. 45C), which is 2.4′ earlier than thesuspected hApo AI standard. To purify the cleavage product from Apo28(oleosin-klip8-pro-Apo AI), fractions were collected from 7 to 25′ at 1mL each. The major polypeptide peak is at 18′ (FIG. 45D), which is 2.4′earlier than the suspected hApo AI standard but similar to the Apo26run.

Mass Spectrometry

Mass spectra were acquired by Doug Olson (National Research Council ofCanada, Plant Biotechnology Institute BioAnalytical Spectroscopy Group,Saskatoon, SK) on an Applied Biosystems Voyager-DE STR matrix assistedlaser desorption ionisation time of flight (MALDI-TOF) mass spectrometerinstrument (Applied Biosystems, Foster City, Calif.). Samples werespotted onto a OPI-TOF LC MALDI insert (Applied Biosystems, Foster City,Calif.) using a matrix of sinapinic acid saturated in 30%acetonitrile/70% water/0.1% TFA. Ions were accelerated at +20 kV andmasses were detected in linear mode, with horse heart myoglobin used asan external calibrant. —Electrospray ionization mass spectrometry of thepurified recombinant mature Apo25 protein gave a molecular mass of 28,325 Da, which is 6 Da greater than calculated molecular weight of 28,319 Da. The difference in value from the observed value from theexpected, may be due to a case of limited sample leading to a decreasedsignal to noise ratio and a decreased accuracy. The expected molecularweight of mature Apo AI is 28, 187 Da, but due to the presence of theadditional Met residue, the cleaved recombinant mature Apo AI proteinhas an increased molecular weight. The purified standard hApo AI proteinwas also analyzed by mass spectrometry, and it possessed two distinctpeaks at 25, 969 Da and 22, 815 Da. Both of these observed values aresignificantly lower than the expected value of 28, 187 Da; however,these two predominant lower molecular weights were previously observedon the immunoblots. It is likely that it is this decrease in molecularweight that results in the slightly different elution profile of thehuman and recombinant proteins as was seen by RP-HPLC.

Example 7 Transformation of Safflower

This transformation protocol is similar to that outlined by OrlilcowskaT. K. et al. ((1995) Plant Cell, Tissue and Organ Culture 40: 85-91),but with modifications and improvements both for transforming S-317 andfor using phosphinothricin as the selectable marker. Decontaminate seedsfrom S-317 California variety of safflower, which are not damaged,cracked or diseased, in 0.1% HCl₂ for 12 minutes followed by 4-5 rinseswith sterile distilled water. Germinate sterile seeds in the dark on MSmedium (Murashige T. & Skoog F (1962) Physiol. Plant. 15: 473-497) with1% sucrose and 0.25% Gelrite. Initiate Agrobacterium cultures fromfrozen glycerol stocks in 5 ml AB minimal liquid media with antibioticselection, and grow for 48 hours at 28° C. Grow an aliquot of thisculture grown overnight in 5 ml of Luria broth with selection fortransformation. Wash 6-8 ml of bacterial cells twice with AB media, andmake up to a final cell density of 0.4-0.5 (OD600).

Remove two-day-old cotyledons from germinated seedlings, dip in theprepared Agrobacterium cells, and plate on MS medium with 3% sucrose, 4μM N6-benzyladenine (BA) and 0.8 μM naphthaleneacetic acid (NAA).Incubate plates at 21° C. under dark conditions. After 3 days, transferto the same medium with 300 mg/L timentin. After an additional 4 days,move all cultures to the light. After 3 days, place explants onselection medium with phosphinothricin added at 0.5 mg/L. For continuedbud elongation, transfer explants weekly onto MS medium withoutphytohormones but with twice the basal amount of KNO₃. Excise shootsthat had elongated to greater than 10 mm from the initial explant andindividually grow on selection. For rooting, place green shoots,representing putative transgenic tissue, on MS medium with 2% sucrose,10 μM indolebutyric acid and 0.5 μM NAA. Transfer rooted shoots to awell drained soil-less mix and grow under high humidity and 12 hours oflight.

Example 8 Flax Transformation Protocol

This transformation procedure is similar to that outlined by Dong J. andMcHughen A. (Plant Cell Reports (1991) 10:555-560), Dong J. and McHughenA. (Plant Sciences (1993) 88:61-71) and Mlynarova et al. (Plant CellReports (1994) 13: 282-285). Decontaminate flax seeds, which are notdamaged, cracked or diseased, in a 70% ethanol solution for 5 to 7minutes, followed by 25 minutes in a 50% bleach solution with Tween 20(3-4 drops per 100 ml) with continuous stirring. Rinse seeds 5 to 7times with sterile distilled water. Germinate decontaminated seeds inthe light on MS medium (Murashige T. & Skoog F (1962) Physiol. Plant.15: 473-497) with 2% sucrose and 0.3% Gelrite® in Magenta jars. Fortransformation, grow Agrobacterium cultures overnight in AB broth plusthe appropriate antibiotic for selection. Wash 6 to 8 ml of overnightcells twice, and re-suspended in 5 ml of AB broth; add 2 ml of thisstock to 98 ml of induction medium (MS basal medium with 3% sucrose, 5μM 6-benzylaminopurine (BA) and 0.25 μM alpha-naphthalene acetic acid(NAA) and adjust for a final OD₆₀₀ of 1.0.

Section hypocotyl explants, and inoculate in the prepared Agrobacteriumcell solution for about 4 h (stir plates gently 1-2 times during thisperiod). After the infection period, remove explants from the liquidinoculation medium and blot on sterile filter paper. Plate 15-20explants on 0.7% agar-solidified induction medium in tissue cultureplates. Seal the plates with plastic wrap, and co-cultivate explants for48 h under light conditions (23-24° C.). After 2 days, transfer thegreen, meristematic explants to the same medium containing 300 mg/LTimentin (pre-selection media) and wrap with plastic wrap. After 3 days,transfer the cultures to the above medium containing 10 mg/L DL PPT(Selection 1). Wrap the plates with Parafilm® and incubate at 24° C.under light conditions. Transfer cultures every two weeks and keep onthis media for one month. For shoot elongation, transfer the culturesevery two weeks on selection medium II (MS basal medium containing 2%sucrose, 500 mg/L MES buffer, 300 mg/L Timentin and 10 mg/L DL PPT) inMagenta jars. Putative transformed shoots, which survived selection, aredark green and form vigorous roots in 7-10 days when plantedindividually on selection II media. Transfer rooted shoots to sterilizedgreenhouse soil mix in small pots and cover plantlets with clear plasticcups for acclimatization. For maturation, transfer actively growingplants to one-gallon pots with a well-drained soil mix and grow undergreenhouse conditions.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

SUMMARY OF SEQUENCES

SEQ ID NO:1 and 2 set forth the nucleotide sequence and the deducedamino acid sequence, respectively, of the human pro-Apo AI protein.

SEQ ID NO:3 and 4 set forth the nucleotide sequence and the deducedamino acid sequence, respectively, of the human Apo AI Milano protein.

SEQ ID NO:5 and 6 set forth the nucleotide sequence and the deducedamino acid sequence, respectively, of the human Apo AI Paris protein.

SEQ ID NO:1, 7 and 8 are known human Apolipoprotein sequences which aredescribed in Table 1.

SEQ ID NO:9-24 are known Apolipoprotein A-I sequences which aredescribed in Table 1.

SEQ ID NO:25-34 are known Apolipoprotein A-IV sequences which aredescribed in Table 1.

SEQ ID NO:35-55 are known Apolipoprotein E sequences which are describedin Table 1.

SEQ ID NO:56 sets forth the nucleic acid sequence of an Arabidopsisthaliana thioredoxin

SEQ ID NO:57 sets forth the nucleic acid sequence of a soluble greenfluorescent protein

SEQ ID NO:58 sets forth the amino acid sequence of a PRS signalsequence.

SEQ ID NO:59-116 are known oleosin oil body protein sequences which aredescribed in Table 3.

SEQ ID NO:117-129 are known caleosin oil body protein sequences whichare described in Table 3.

SEQ ID NO:130-137 are known steroleosin oil body protein sequences whichare described in Table 3.

SEQ ID NO:138 sets forth a known Arabidopsis thaliana oleosin oil bodyprotein sequence.

SEQ ID NO:139 sets forth a known Brassica napus oleosin oil body proteinsequence.

SEQ ID NO:140 sets forth a known Arabidopsis thaliana caleosin oil bodyprotein nucleic acid sequence.

SEQ ID NO:141 sets forth a known Arabidopsis thaliana caleosin oil bodyprotein nucleic acid sequence.

SEQ ID NO:142 sets forth a known stereoleosin oil body protein nucleicacid sequence.

SEQ ID NO:143 sets forth the nucleotide sequence for the klip8 cleavagesequence.

SEQ ID NO:144 and 145 set forth the nucleotide sequence and the deducedamino acid sequence, respectively, of the Apo10 clone.

SEQ ID NO:146 sets forth the nucleotide sequence of the forward primer1186 which is complementary to the 5′ region of GFP and is designed toremove the NcoI site.

SEQ ID NO:147 sets forth the nucleotide sequence of the reverse primer1187 which is complementary to the 3′ region of GFP and is designed toadd PstI, XbaI and HindIII sites after the stop codon.

SEQ ID NO:148 sets forth the nucleotide sequence of the forward primer1190 which is complementary to the 5′ region of mature Apo AI and isdesigned to add a NcoI site to the start of the gene.

SEQ ID NO:149 sets forth the nucleotide sequence of the reverse primer1189 which is complementary to the 5′ region of mature Apo AI and isdesigned to remove the stop codon of the gene and add a BamHI site toassist in creating an in-frame translational fusion with GFP.

SEQ ID NO:150 and 151 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo11 clone.

SEQ ID NO:152 sets forth the nucleotide sequence of the forward primer1191 which is complementary to the 5′ region of pro-Apo AI and isdesigned to add a NcoI site to the start of the gene.

SEQ ID NO:153 and 154 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo12 clone.

SEQ ID NO:155 and 156 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo13 clone.

SEQ ID NO:157 and 158 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo15 clone.

SEQ ID NO:159 and 160 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo16 clone.

SEQ ID NO:161 and 162 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo17 clone.

SEQ ID NO:163 and 164 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo18 clone.

SEQ ID NO:165 and 166 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo19 clone.

SEQ ID NO:167 sets forth the nucleotide sequence for forward primer 1177which is complementary to the 5′ region of PRS/Apo AI (clone Apo15) andis designed to amplify the start of the plant presequence (PRS) whichcontains a BspHI site at the start codon.

SEQ ID NO:168 sets forth the nucleotide sequence for reverse primer 1178which is complementant to the 3′ region of Apo AI and is designed toremove the stop codon of the gene and add a BamHI site to assist increating an in-frame translational fusion with GFP.

SEQ ID NO:169 and 170 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo20 clone.

SEQ ID NO:171 and 172 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo21 clone.

SEQ ID NO:173 sets forth the nucleotide sequence of forward primer 1203which is complementary to the 5′ region of Apo AI and is designed to adda NcoI site to the start of mature Apo AI.

SEQ ID NO:174 sets forth the nucleotide sequence of reverse primer 1206which is complementary to the 3′ region of Apo AI and is designed to adda HindIII site after the stop codon.

SEQ ID NO:175 and 176 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo22 clone.

SEQ ID NO:177 sets forth the nucleotide sequence of forward primer 1201which is complementary to the 5′ region of pro AI and is designed to addan NcoI site to the start of pro-Apo AI.

SEQ ID NO:178 and 179 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo23 clone.

SEQ ID NO:180 and 181 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo24 clone.

SEQ ID NO:182 and 183 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo25 clone.

SEQ ID NO:184 and 185 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo26 clone.

SEQ ID NO:186 and 187 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo27 clone.

SEQ ID NO:188 sets forth the nucleotide sequence of forward primer 1200which is complementary to the 5′ region of mature Apo AI and is designto add an XhoI site and extra nucleotides to facilitate in-frame cloninginto the klip8 cleavage sequence to the start of pro-Apo AI.

SEQ ID NO:189 and 190 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo27M clone.

SEQ ID NO:191 sets forth the nucleotide sequence of forward primer 1202which is complementary to the 5′ region of the human Apo AI and isdesigned to amplify the human Apo AI sequence, add a XhoI site and extranucleotides to facilitate in-frame cloning into the klip8 cleavagesequence to the start of mat-Apo AI.

SEQ ID NO:192 sets forth the nucleotide sequence of forward primer 1225is a blunt ended primer which makes a base pair mutation from C to T tochange an Arg residue into a Cys residue thereby creating the Apo-Milanomutation.

SEQ ID NO:193 and 194 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo28 clone.

SEQ ID NO:195 sets forth the nucleotide sequence of forward primer 1205which is complementary to the 5′ region of pro-Apo AII and is designedto be a blunt ended primer which adds a silent mutation to remove thefirst XhoI site.

SEQ ID NO:196 and 197 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo29 clone.

SEQ ID NO:198 and 199 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo30 clone.

SEQ ID NO:200 and 201 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo31 clone.

SEQ ID NO:202 and 203 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo32 clone.

SEQ ID NO:204 and 205 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo33 clone.

SEQ ID NO:206 and 207 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo34 clone.

SEQ ID NO:208 and 209 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo35 clone.

SEQ ID NO:210 sets forth the nucleotide sequence of reverse primer 1208which is complementary to the 3′ region of pro-Apo AI and is designed toadd a KDEL sequence before the stop codon and a HindIII site after thestop codon.

SEQ ID NO:211 and 212 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo36 clone.

SEQ ID NO:213 and 214 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo37 clone.

SEQ ID NO:215 and 216 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo38 clone.

SEQ ID NO:217 and 218 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo39 clone.

SEQ ID NO:219 sets forth the nucleotide sequence of forward primer 1207which is complementary to the 5′ region of the klip8 cleavage sequenceand is designed to amplifies the start of the klip8 sequence and adds aSalI site to the start codon.

SEQ ID NO:220 and 221 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo40 clone.

SEQ ID NO:222 and 223 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo41 clone.

SEQ ID NO:224 and 225 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo42 clone.

SEQ ID NO:226 and 227 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo43 clone.

SEQ ID NO:228 and 229 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo44 clone.

SEQ ID NO:230 and 231 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo45 clone.

SEQ ID NO:232 and 233 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo46 clone.

SEQ ID NO:234 and 235 set forth the nucleotide and deduced amino acidsequences, respectively, of the Apo47 clone.

SEQ ID NO:236 sets forth the nucleotide sequence of forward primer 1226which is complementary to the 5′ region of the maize oleosin sequenceand is designed to amplifies the maize oleosin sequence and adds a NcoIsite to the start codon.

SEQ ID NO:237 sets forth the nucleotide sequence of forward primer 1227which is complementary to the 3′ region of the maize oleosin sequenceand is designed to amplify the maize oleosin, remove the stop codon ofthe gene and add a HindIII site to assist in creating an in-frametranslational fusion with klip8/matApo AI.

SEQ ID NO:238 sets forth the nucleotide sequence of forward primer 1228which is complementary to the 5′ region of the Apo25 clone and isdesigned to amplify the Apo25 sequence and adds a SalI site to the startcodon.

SEQ ID NO:239 sets forth the nucleotide sequence of reverse primer 1229which is complementary to the 3′ region of the Apo25 clone and isdesigned to amplify the Apo25 sequence and adds a BamHI site after thestop codon.

SEQ ID NO:240 sets forth the amino acid sequence of the single chainantibody D9scFv.

SEQ ID NOS. 241-251 are known Apolipoprotein AV sequences which aredescribed in Table 1.

TABLE 1 Examples of known Apolipoprotein sequences SEQUENCE ID. NO.APOLIPOPROTEIN SOURCE (Accession number) Apolipoprotein A-I 1 Human (NM000039, BC005380, J00098, M11791, M27875, M29068, X00566, X01038,X02162, X07496) 9 Danio rerio (NP_571203) 10 Rattus norvegicus (P04639)11 Bos taurus (P15497) 12 Mus musculus (Q00623) 13 Ovis aries (AAB57840)14 Sus scrofa (P18648) 15 Cyprinus carpio (CAC34942) 16 Gallus gallus(AAA48593) 17 Oryctolagus cuniculus (P09809) 18 Macaca fascicularis(P15568) 19 Coturnix japonica (P32918) 20 Canis familiaris (P02648) 21Tupaia belangeri (O18759) 22 Anas platyrhynchos (O42296) 23 Papio anubis(AAA35380) 24 Macaca mulatto (P14417) Apolipoprotein A-IV 7 Human (NM0000482, J02758, M10373, M13654, M14566, M14642, X13629, P0672) 25Rattus norvegicus (AAA85909) 26 Mus musculus (P06728) 27 Mus musculuscastaneus (AAA37216) 28 Sus scrofa (O46409) 29 Papio anubis (Q28758) 30Macaca fascicularis (P33621) 31 Pan troglodytes (I54248) 32 Papio sp.(A47141) 33 Gillichthys mirabilis (AAG13299) 34 Oryctolagus cuniculus(AAB34783) Apolipoprotein A-V 241 Mus musculus (NM_080434) 242 Rattusnorvegicus (NM_080576) 243 Homo sapiens (NP_443200) 244 Mus musculus(BC011198) 245 Homo sapiens (AY555191) 246 Homo sapiens (AY422949) 247Mus musculus (AF327059) 248 Homo sapiens (AF202890) 249 Homo sapiens(AF202889) 250 Rattus norvegicus (AF202888) 251 Rattus norvegicus(AF202887) Apolipoprotein E 8 Human (NM 000041, AF050154, AF261279,BC003557, K00396, M10065, M12529, X00199, X92000, Z70760) 35 Rattusnorvegicus (P02650) 36 Danio rerio (O42364) 37 Bos Taurus (Q03247) 38Mus musculus (P08226) 39 Canis familiaris (P18649) 40 Saimiri sciureus(Q28995) 41 Macaca mulatto (Q28502) 42 Sus scrofa (P18650) 43Oryctolagus cuniculus (P18287) 44 Papio anubis (P05770) 45 Macacafascicularis (P10517) 46 Cavia porcellus (P23529) 47 Zalophuscalifornianus (JC5566) 48 Ovis sp. (JC6549) 49 Pongo pygmaeus (AAG28580)50 Hylobates lar (AAG28581) 51 Gorilla gorilla (AAG28579) 52 Pantroglodytes (AAG28578) 53 Tupaia glis (AAG21401) 54 Oncorhynchus mykiss(CAB65320) 55 Scophthalmus maximus (CAB65356)

TABLE 2 Examples of known apolipoprotein mutations and modificationsProtein Mutation Reference POINT MUTATIONS Apo AI Glu198Lys Strobl W etal., Pediatr Res. August 1988; 24(2):222-8 Apo AI Gly26Arg Vigushin DMet al. Q J Med. March 1994; 87(3):149-54 Apo AI Leu60Arg Soutar AK etal. Proc Natl Acad Sci U S A. Aug. 15, 1992; 89(16):7389-93 Apo AIVal156Glu Cho KH and Jonas A. J Biol Chem. Sep. 1, 2000; 275(35):26821-7Apo AI Baltimore Arg10Leu Ladias JA et al., Hum Genet. April 1990;84(5):439-45 Apo AI Giessen Pro143Arg Utermann G et al., Eur J Biochem.Oct. 15, 1984; 144(2):325-31 Apo AI Fukuoka Glu110Lys Takada Y et al.,Biochim Biophys Acta. Apr. 2, 1990; 1043(2):169-76. Apo AI Fin Arg159LeuMiettinen HE et al. Arterioscler Thromb Vasc Biol. January 1997;17(1):83-90 Apo AI Milano Arg173Cys Cheung MC et al., Biochim BiophysActa. May 2, 1988; 960(1):73-82 Apo AI Paris Arg151Cys Bruckert E etal., Atherosclerosis. Jan. 3, 1997; 128(1):121-8 Apo AV Val153MetHubacek et al. Physiol. Res. 2004. Cys185Gly 53:225-228 Apo AV Thr131CysHubacek et al. Clin. Genet. 2004. 65: Ser19Trp 126-130 Apo E Arg136CysHubacek JA et al. Physiol Res. 2002; 51(1):107-8 Apo E*5 Gln204Lys,Scacchi R. et al. Hum Biol. Cys112Arg, or April 2003; 75(2):293-300;Feussner et al., J Glu212Lys Lipid Res. August 1996; 37(8):1632-45 ApoE1 Lys146Glu Mann WA et al., J Clin Invest. August 1995; 96(2):1100-7Apo E2 Arg136Cys Feussner G. et al. Eur J Clin Invest. January 1996;26(1):13-23 Apo E2 Arg142Leu Richard P et al. Atherosclerosis. Jan. 6,1995; 112(1):19-28 Apo E2 Arg25Cys Matsunaga et al. Kidney Int. August1999; 56(2):421-7 Apo E2 Lys146Gln Smit M et al., j Lipid Res. January1990; 31(1):45-53 Apo E2 Arg136Ser Wardell MR et al. J Clin Invest.Christchurch August 1987; 80(2):483-90. Apo E3 Arg136Cys Walden CC etal. J Clin Endocrinol Metab. March 1994; 78(3):699-704 Apo E3 Arg136HisMinnich A et al. J Lipid Res. January 1995; 36(1):57-66. ApoE5-Frankfurt Gln81Lys, Cys112Arg Ruzicka V et al. Electrophoresis.October 1993; 14(10):1032-7 DELETION MUTATIONS Apo AI nichinan G1u235deletion Han et al. Arterioscler Thromb Vasc Biol. June 1999;19(6):1447-55 Apo AI Lys 107 deletion Amarzguioui M et el. BiochemBiophys Res Commun. Jan. 26, 1998; 242(3):534-9 FRAMESHIFT MUTATIONS ApoAI Sasebo partial gene Moriyama K et al. Arterioscler duplication,tandem Thromb Vasc Biol. repeat of bases 333 to December 1996;16(12):1416-23 355 from the 5′ end of exon 4 resulting with prematuretermination after amino acid 207 CHEMICAL MODIFICATIONS Apo AIsulfoxidized Met-112 Jonas A et al. Biochim Biophys Acta. and Met-148residues Feb. 24, 1993; 1166(2-3):202-10 and the corresponding reducedform

TABLE 3 Examples of known oil body protein sequences SEQ. ID Oil BodyProtein Motif (Amino Acid Sequence NO. Identifier) {Nucleic AcidSequence Identifier} Oleosin 59 (A84654) Arabidopsis thaliana probableoleosin 60 (AAA87295) Arabidopsis thaliana oleosin {Gene L40954} 61(AAC42242) Arabidopsis thaliana oleosin {Gene AC005395} 62 (AAF01542)Arabidopsis thaliana putative oleosin {Gene AC009325} 63 (AAF69712)Arabidopsis thaliana F27J15.22 {Gene AC016041} 64 (AAK96731) Arabidopsisthaliana oleosin-like protein {Gene AY054540} 65 (AAL14385) Arabidopsisthaliana AT5g40420/MPO12_130 oleosin isoform {Gene AY057590} 66(AAL24418) Arabidopsis thaliana putative oleosin {Gene AY059936} 67(AAL47366) Arabidopsis thaliana oleosin-like protein {Gene AY064657} 68(AAM10217) Arabidopsis thaliana putative oleosin {Gene AY081655} 69(AAM47319) Arabidopsis thaliana AT5g40420/MPO12_130 oleosin isoform{Gene AY113011} 70 (AAM63098) Arabidopsis thaliana oleosin isoform {GeneAY085886} 71 (AAO22633) Arabidopsis thaliana putative oleosin {GeneBT002813} 72 (AAO22794) Arabidopsis thaliana putative oleosin protein{Gene BT002985} 73 (AAO42120) Arabidopsis thaliana putative oleosin{Gene BT004094} 74 (AAO50491) Arabidopsis thaliana putative oleosin{Gene BT004958} 75 (AAO63989) Arabidopsis thaliana putative oleosin{Gene BT005569} 76 (AAQ56108) Arabidopsis lyrata subsp. Lyrata Oleosin.{Gene AY292860} 77 (BAA97384) Arabidopsis thaliana oleosin-like {GeneAB023044} 78 (BAB02690) Arabidopsis thaliana oleosin-like protein {GeneAB018114} 79 (BAB11599) Arabidopsis thaliana oleosin, isoform 21K {GeneAB006702} 80 (BAC42839) Arabidopsis thaliana putative oleosin protein{Gene AK118217} 81 (CAA44225) Arabidopsis thaliana oleosin {Gene X62353}82 (CAA63011) Arabidopsis thaliana oleosin, type 4 {Gene X91918} 83(CAA63022) Arabidopsis thaliana oleosin, type 2 {Gene X91956} 84(CAA90877) Arabidopsis thaliana oleosin {Gene Z54164} 85 (CAA90878)Arabidopsis thaliana oleosin {Gene Z54165} 86 (CAB36756) Arabidopsisthaliana oleosin, 18.5 K {Gene AL035523} 87 (CAB79423) Arabidopsisthaliana oleosin, 18.5 K {Gene AL161562} 88 (CAB87945) Arabidopsisthaliana oleosin-like protein {Gene AL163912} 89 (P29525) Arabidopsisthaliana oleosin 18.5 kDa {Gene X62353, CAA44225, AL035523, CAB36756,CAB36756, CAB79423, Z17738, S22538} 90 (Q39165) Arabidopsis thalianaOleosin 21.2 kDa (Oleosin type 2). {Gene L40954, AAA87295, X91956,CAA63022, Z17657, AB006702, BAB11599, AY057590, AAL14385, S71253 91(Q42431) Arabidopsis thaliana Oleosin 20.3 kDa (Oleosin type 4) {GeneZ54164, CAA90877, X91918, CAA63011, AB018114, BAB02690, AY054540,AAK96731, AY064657, AAL47366, AY085886, AAM63098, Z27260, Z29859, S7128692 (Q43284) Arabidopsis thaliana Oleosin 14.9 kDa. {Gene Z54165,CAA90878, AB023044, BAA97384, Z27008, CAA81561} 93 (S22538) Arabidopsisthaliana oleosin, 18.5 K 94 (S71253) Arabidopsis thaliana oleosin, 21 K95 (S71286) Arabidopsis thaliana oleosin, 20 K 96 (T49895) Arabidopsisthaliana oleosin-like protein 97 (AAB22218) Brassica napus oleosin napII98 (AAD24547) Brassica oleracea oleosin 99 (CAA43941) Brassica napusoleosin BN-III {Gene X63779} 100 (CAA45313) Brassica napus oleosin BN-V{Gene X63779} 101 (P29109) Brassica napus Oleosin Bn-V (BnV) {GeneX63779, CAA45313, S25089) 102 (P29110) Brassica napus Oleosin Bn-III(BnIII) {Gene X61937, CAA43941, S22475) 103 (P29111) Brassica napusMajor oleosin NAP-II {Gene X58000, CAA41064, S70915) 104 (S22475)Brassica napus oleosin BN-III 105 (S50195) Brassica napus Oleosin 106(T08134) Brassica napus Oleosin-like 107 (AAB01098) Daucus carotaoleosin 108 (T14307) carrot oleosin 109 (A35040) Zea mays oleosin 18 110(AAA67699)Zea mays oleosin KD18 {Gene J05212} 111 (AAA68065) Zea mays 16kDa oleosin {Gene U13701} 112 (AAA68066) Zea mays 17 kDa oleosin {GeneU13702} 113 (P13436) Zea mays OLEOSIN ZM-I (OLEOSIN 16 KD) (LIPIDBODY-ASSOCIATED MAJOR PROTEIN) {Gene U13701, AAA68065, M17225, AAA33481,A29788} 114 (P21641) Zea mays Oleosin Zm-II (Oleosin 18 kDa) (Lipidbody-associated protein L2) {Gene 105212, AAA67699, A35040} 115 (S52029)Zea mays oleosin 16 116 (S52030) Zea mays oleosin 17 Caleosin 117(XP_467656) putative caleosin [Oryza sativa (japonica cultivar- group)].118 (BAD16161) putative caleosin [Oryza sativa (japonica cultivar-group)]. {Gene AP005319} 119 (NP_973892) caleosin-related family protein[Arabidopsis thaliana]. {Gene NM_202163} 120 (NP_564996)caleosin-related family protein [Arabidopsis thaliana]. {Gene NM_105736}121 (NP_564995) caleosin-related family protein [Arabidopsis thaliana]{Gene NM_105735} 122 (NP_200335) caleosin-related familyprotein/embryo-specific protein, putative [Arabidopsis thaliana]. {GeneNM_124906} 123 (NP_173739) caleosin-related [Arabidopsis thaliana].{Gene NM_102174} 124 (NP_173738) caleosin-related family protein[Arabidopsis thaliana] {Gene NM_102173} 125 (AAQ74240) caleosin 2[Hordeum vulgare]. {Gene AY370892} 126 (AAQ74239) caleosin 2 [Hordeumvulgare]. {Gene AY370891} 127 (AAQ74238) caleosin 1 [Hordeum vulgare].{Gene AY370890} 128 (AAQ74237) caleosin 1 [Hordeum vulgare]. {GeneAY370889} 129 (AAF13743) caleosin [Sesamum indicum]. {Gene AF109921}Steroleosin 130 (XP_465935) putative steroleosin [Oryza sativa (japonicacultivar-group)]. {Gene XM_465935} 131 (XP_465933) putative steroleosin[Oryza sativa (japonica cultivar-group)]. {Gene XM_465933} 132(AAT77030) putative steroleosin-B [Oryza sativa (japonicacultivar-group)]. {Gene AC096856} 133 (BAD23084) putative steroleosin[Oryza sativa (japonica cultivar-group)] {Gene AP004861} 134 (BAD23082)putative steroleosin [Oryza sativa (japonica cultivar-group)] {GeneAP004861} 135 (AAM46847) steroleosin-B [Sesamum indicum]. {GeneAF498264} 136 (AAL13315) steroleosin [Sesamuin indicum]. {Gene AF421889}137 (AAL09328) steroleosin [Sesamum indicum]. {Gene AF302806}

1-38. (canceled)
 39. A composition comprising substantially pure oilbodies comprising apolipoprotein obtained from plants.
 40. A nucleicacid sequence encoding apolipoprotein linked to nucleic acid sequencecomprising a nucleic acid capable of controlling expression in a plantcell.
 41. A nucleic acid sequence according to claim 40 wherein saidplant cell is a seed cell.
 42. A nucleic acid sequence according toclaim 41 wherein said nucleic acid sequence capable of controllingexpression in a plant cell is a seed-preferred promoter.
 43. A nucleicacid according to claim 42 wherein said seed-preferred promoter is thephaseolin promoter.
 44. A nucleic acid sequence according to claim 41wherein said nucleic acid sequence capable of controlling expression inplant seeds is a constitutive promoter.
 45. A nucleic acid sequenceaccording to claim 44 wherein said promoter is the ubiquitin promoter.46. A recombinant expression vector suitable for expression in a plantcell comprising a nucleic acid sequence encoding an apolipoproteinpolypeptide.
 47. A method for preparing substantially pureapolipoprotein comprising: (a) providing a chimeric nucleic acidconstruct comprising in the 5′ to 3′ direction of transcription asoperably linked components: (i) a nucleic acid sequence capable ofcontrolling expression in plant seed cells; and (ii) a nucleic acidsequence encoding an apolipoprotein polypeptide; (b) introducing thechimeric nucleic acid construct into a plant cell; (c) growing the plantcell into a mature plant; and (d) obtaining seed from said plant whereinthe seed comprises apolipoprotein; and (e) separating apolipoproteinfrom the plant seed constituents to obtain substantially pureapolipoprotein.
 48. (canceled)
 49. A composition comprisingsubstantially pure apolipoprotein obtained from a plant.